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The reverse transcriptase (RT) model of immunoglobulin (Ig) somatic hypermutation (SHM) has received insufficient scientific attention. This is understandable given that DNA deamination mediated by activation-induced deaminase (AID), the initiating step of Ig SHM, has dominated experiments since 2002. We summarise some key history of the RT Ig SHM model dating to 1987. For example, it is now established that DNA polymerase η, the sole DNA repair polymerase involved in post-replication short-patch repair, is an efficient cellular RT. This implies that it is potentially able to initiate target site reverse transcription by RNA-directed DNA repair at AID-induced lesions. Recently, DNA polymerase θ has also been shown to be an efficient cellular RT. Since DNA polymerase θ plays no significant role in Ig SHM, it could serve a similar RNA-dependent DNA polymerase role as DNA polymerase η at non- Ig loci in the putative RNA-templated nucleotide excision repair of bulky adducts and other mutagenic lesions on the transcribed strand. A major yet still poorly recognised consequence of the proposed RT process in Ig SHM is the generation of significant and characteristic strand-biased mutation signatures at both deoxyadenosine/deoxythymidine and deoxyguanosine/deoxycytidine base pairs. In this historical perspective, we highlight how diagnostic strand-biased mutation signatures are detected in vivo during SHM at both Ig loci in germinal centre B lymphocytes and non- Ig loci in cancer genomes. These strand-biased signatures have been significantly obscured by technical issues created by improper use of the polymerase chain reaction technique. A heightened awareness of this fact should contribute to better data interpretation and somatic mutation pattern recognition both at Ig and non- Ig loci. • Clear strand biased patterns at A/T and G/C base pairs evident in immunoglobulin somatic hypermutation (Ig SHM). • These strand biases are readily explained by a reverse transcription mechanism of Ig SHM. • Theses patterns in the past have been obscured by the PCR technique used to harvest diverse related Ig sequences. • Strand biased mutation patterns are evident at both Ig loci in B lymphocytes and non- Ig loci in cancer genomes. [ABSTRACT FROM AUTHOR]

• Hepatocellular carcinomas (HCCs) occur with highest frequency in East Asia. • HCCs are associated with aflatoxin exposures and chronic inflammation. • NEIL1 initiates repair of aflatoxin- and inflammation-induced DNA base damage. • Variants of NEIL1 that occur in East Asian populations are characterized. • A subset of NEIL1 variants are expected to have human health implications. The combination of chronic dietary exposure to the fungal toxin, aflatoxin B 1 (AFB 1), and hepatitis B viral (HBV) infection is associated with an increased risk for early onset hepatocellular carcinomas (HCCs). An in-depth knowledge of the mechanisms driving carcinogenesis is critical for the identification of genetic risk factors affecting the susceptibility of individuals who are HBV infected and AFB 1 exposed. AFB 1 -induced mutagenesis is characterized by G to T transversions. Hence, the DNA repair pathways that function on AFB 1 -induced DNA adducts or base damage from HBV-induced inflammation are anticipated to have a strong role in limiting carcinogenesis. These pathways define the mutagenic burden in the target tissues and ultimately limit cellular progression to cancer. Murine data have demonstrated that NEIL1 in the DNA base excision repair pathway was significantly more important than nucleotide excision repair relative to elevated risk for induction of HCCs. These data suggest that deficiencies in NEIL1 could contribute to the initiation of HCCs in humans. To investigate this hypothesis, publicly-available data on variant alleles of NEIL1 were analyzed and compared with genome sequencing data from HCC tissues derived from individuals residing in Qidong County (China). Three variant alleles were identified and the corresponding A51V, P68H, and G245R enzymes were characterized for glycosylase activity on genomic DNA containing a spectrum of oxidatively-induced base damage and an oligodeoxynucleotide containing a site-specific AFB 1 -formamidopyrimidine guanine adduct. Although the efficiency of the P68H variant was modestly decreased, the A51V and G245R variants showed nearly wild-type activities. Consistent with biochemical findings, molecular modeling of these variants demonstrated only slight local structural alterations. However, A51V was highly temperature sensitive suggesting that its biological activity would be greatly reduced. Overall, these studies have direct human health relevance pertaining to genetic risk factors and biochemical pathways previously not recognized as germane to induction of HCCs. [ABSTRACT FROM AUTHOR]

Nei-like glycosylase 1 (NEIL1) is a DNA repair enzyme that initiates the base excision repair (BER) pathway to cleanse the human genome of damage. The substrate specificity of NEIL1 includes several common base modifications formed under oxidative stress conditions, as well as the imidazole ring open adducts that are induced by alkylating agents following initial modification at N7 guanine. An example of the latter is the persistent and mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B 1 (AFB 1 -FapyGua) adduct, resulting from the alkylating agent aflatoxin B 1 (AFB 1) exo -8–9-epoxide. Naturally occurring single nucleotide polymorphic (SNP) variants of NEIL1 are hypothesized to be associated with an increased risk for development of early-onset hepatocellular carcinoma (HCC), especially in environments with high exposures to aflatoxins and chronic inflammation from viral infections and alcohol consumption. Given that AFB 1 exposures and hepatitis B viral (HBV) infections represent a major problem in the developing countries of sub-Saharan Africa, it is pertinent to study SNP NEIL1 variants that are present in this geographic region. In this investigation, we characterized the three most common NEIL1 variants found in this region: P321A, R323G, and I182M. Biochemical analyses were conducted to determine the proficiencies of these variants in initiating the repair of DNA lesions. Our data show that damage recognition and excision activities of P321A and R323G were near that of wild-type (WT) NEIL1 for both thymine glycol (ThyGly) and AFB 1 -FapyGua. The substrate specificities of these variants with respect to various oxidatively-induced base lesions were also similar to that of WT. In contrast, the I182M variant was unstable, such that it precipitated under a variety of conditions and underwent rapid inactivation at a biologically relevant temperature, with partial stabilization being observed in the presence of undamaged DNA. This study provides insight regarding the potential increased risk for early-onset HCC in human populations carrying the NEIL1 I182M variant. • Aflatoxin exposure and HBV infection, the known risk factors for hepatocellular carcinoma, are common in sub-Saharan Africa. • Single nucleotide polymorphic variants of NEIL1 in sub-Saharan Africa populations were characterized. • DNA substrates included aflatoxin-derived lesions and oxidatively-induced damage to model environmental exposures. • The P321A and R323G variants exhibited activities comparable to wild-type NEIL1. • The I182M variant was temperature sensitive but was partially stabilized by undamaged DNA. [ABSTRACT FROM AUTHOR]

DNA adducts and strand breaks are induced by various exogenous and endogenous agents. Accumulation of DNA damage is implicated in many disease processes, including cancer, aging, and neurodegeneration. The continuous acquisition of DNA damage from exogenous and endogenous stressors coupled with defects in DNA repair pathways contribute to the accumulation of DNA damage within the genome and genomic instability. While mutational burden offers some insight into the level of DNA damage a cell may have experienced and subsequently repaired, it does not quantify DNA adducts and strand breaks. Mutational burden also infers the identity of the DNA damage. With advances in DNA adduct detection and quantification methods, there is an opportunity to identify DNA adducts driving mutagenesis and correlate with a known exposome. However, most DNA adduct detection methods require isolation or separation of the DNA and its adducts from the context of the nuclei. Mass spectrometry, comet assays, and other techniques precisely quantify lesion types but lose the nuclear context and even tissue context of the DNA damage. The growth in spatial analysis technologies offers a novel opportunity to leverage DNA damage detection with nuclear and tissue context. However, we lack a wealth of techniques capable of detecting DNA damage in situ. Here, we review the limited existing in situ DNA damage detection methods and examine their potential to offer spatial analysis of DNA adducts in tumors or other tissues. We also offer a perspective on the need for spatial analysis of DNA damage in situ and highlight Repair Assisted Damage Detection (RADD) as an in situ DNA adduct technique with the potential to integrate with spatial analysis and the challenges to be addressed. • DNA adducts are biomarkers of exogenous and endogenous exposures. • Spatial analysis should be extended to DNA adducts to understand their biological context. • Fluorescence-based methods offer new opportunities for spatial analysis of DNA adducts. • Measuring DNA adducts and their spatial context may provide new insight into changes in DNA repair. [ABSTRACT FROM AUTHOR]

Abstract Chronic dietary exposure to aflatoxin B 1 (AFB 1), concomitant with hepatitis B infection is associated with a significant increased risk for hepatocellular carcinomas (HCCs) in people living in Southeast Asia and sub-Saharan Africa. Human exposures to AFB 1 occur through the consumption of foods that are contaminated with pervasive molds, including Aspergillus flavus. Even though dietary exposures to aflatoxins constitute the second largest global environmental risk factor for cancer development, there are still significant questions concerning the molecular mechanisms driving carcinogenesis and what factors may modulate an individual's risk for HCC. The objective of this review is to summarize key discoveries that established the association of chronic inflammation (most commonly associated with hepatitis B viral (HBV) infection) and environmental exposures to aflatoxin with increased HCC risk. Special emphasis will be given to recent investigations that have: 1) refined the aflatoxin-associated mutagenic signature, 2) expanded the DNA repair mechanisms that limit mutagenesis via adduct removal prior to replication-induced mutagenesis, 3) implicated a specific DNA polymerase in the error-prone bypass and resulting mutagenesis, and 4) identified human polymorphic variants that may modulate individual susceptibility to aflatoxin-induced cancers. Collectively, these investigations revealed that specific sequence contexts are differentially resistant against, or prone to, aflatoxin-induced mutagenesis and that these associations are remarkably similar between in vitro and in vivo analyses. These recent investigations also established DNA polymerase ζ as the major polymerase that confers the G to T transversion signature. Additionally, although the nucleotide excision repair (NER) pathway has been previously shown to repair aflatoxin-induced DNA adducts, recent murine data demonstrated that NEIL1-initiated base excision repair was significantly more important than NER relative to the removal of the highly mutagenic AFB 1 -Fapy-dG adducts. These data suggest that inactivating polymorphic variants of NEIL1 could be a potential driver of HCCs in aflatoxin-exposed populations. [ABSTRACT FROM AUTHOR]

Environmental exposures, reactive by-products of cellular metabolism, and spontaneous deamination events result in a spectrum of DNA adducts that if un-repaired threaten genomic integrity by inducing mutations, increasing instability, and contributing to the initiation and progression of cancer. Assessment of DNA adducts in cells and tissues is critical for genotoxic and carcinogenic evaluation of chemical exposure and may provide insight into the etiology of cancer. Numerous methods to characterize the formation of DNA adducts and their retention for risk assessment have been developed. However, there are still significant drawbacks to the implementation and wide-spread use of these methods, because they often require a substantial amount of biological sample, highly specialized expertise and equipment, and depending on technique, may be limited to the detection and quantification of only a handful of DNA adducts at a time. There is a pressing need for high throughput, easy to implement assays that can assess a broad spectrum of DNA lesions, allowing for faster evaluation of chemical exposures and assessment of the retention of adducts in biological samples. Here, we describe a new methodology, Repair Assisted Damage Detection (RADD), which utilizes a DNA damage processing repair enzyme cocktail to detect and modify sites of DNA damage for a subsequent gap filling reaction that labels the DNA damage sites. This ability to detect and label a broad spectrum of DNA lesions within cells, offers a novel and easy to use tool for assessing levels of DNA damage in cells that have been exposed to environmental agents or have natural variations in DNA repair capacity. [ABSTRACT FROM AUTHOR]

Tyrosyl-DNA phosphodiesterase 1 (TDP1) can remove a wide variety of 3′ and 5′ terminal DNA adducts. Genetic studies in yeast identified TDP1 as a regulator of non-homologous end joining (NHEJ) fidelity in the repair of double-strand breaks (DSBs) lacking terminal adducts. In this communication, we show that TDP1 plays an important role in joining cohesive DSBs in human cells. To investigate the role of TDP1 in NHEJ in live human cells we used CRISPR/cas9 to produce TDP1-knockout (TDP1-KO) HEK-293 cells. As expected, human TDP1-KO cells were highly sensitive to topoisomerase poisons and ionizing radiation. Using a chromosomally-integrated NHEJ reporter substrate to compare end joining between wild type and TDP1-KO cells, we found that TDP1-KO cells have a 5-fold reduced ability to repair I-Sc eI-generated DSBs. Extracts prepared from TDP1-KO cells had reduced NHEJ activity in vitro, as compared to extracts from wild type cells. Analysis of end-joining junctions showed that TDP1 deficiency reduced end-joining fidelity, with a significant increase in insertion events, similar to previous observations in yeast. It has been reported that phosphorylation of TDP1 serine 81 (TDP1-S81) by ATM and DNA-PK stabilizes TDP1 and recruits TDP1 to sites of DNA damage. We found that end joining in TDP1-KO cells was partially restored by the non-phosphorylatable mutant TDP1-S81A, but not by the phosphomimetic TDP1-S81E. We previously reported that TDP1 physically interacted with XLF. In this study, we found that XLF binding by TDP1 was reduced 2-fold by the S81A mutation, and 10-fold by the S81E phosphomimetic mutation. Our results demonstrate a novel role for TDP1 in NHEJ in human cells. We hypothesize that TDP1 participation in human NHEJ is mediated by interaction with XLF, and that TDP1-XLF interactions and subsequent NHEJ events are regulated by phosphorylation of TDP1-S81. [ABSTRACT FROM AUTHOR]

Alkylating agents are genotoxic chemicals that can induce and treat various types of cancer. This occurs through covalent bonding with cellular macromolecules, in particular DNA, leading to the loss of functional integrity under the persistence of modifications upon replication. O 6-alkylguanine (O 6-AlkylG) adducts are proposed to be the most potent DNA lesions induced by alkylating agents. If not repaired correctly, these adducts can result, at the molecular level, in DNA point mutations, chromosome aberrations, recombination, crosslinking, and single- and double-strand breaks (SSB/DSBs). At the cellular level, these lesions can result in malignant transformation, senescence, or cell death. O 6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein capable of removing the alkyl groups from O 6-AlkylG adducts in a damage reversal process that can prevent the adverse biological effects of DNA damage caused by guanine O 6-alkylation. MGMT can thereby defend normal cells against tumor initiation, however it can also protect tumor cells against the beneficial effects of chemotherapy. Hence, MGMT can play an important role in both the prevention and treatment of cancer; thus, it can be considered as a double-edged sword. From a clinical perspective, MGMT is a therapeutic target, and it is important to explore the rational development of its clinical exploitation. [Display omitted] • The role of MGMT in the protection of tumor initiation was summarized. • The role of MGMT in tumor resistance was also described. • The contradictory effects of MGMT were analyzed and discussed. • Future clinical exploration of MGMT was proposed in three aspects. [ABSTRACT FROM AUTHOR]

The apurinic/apyrimidinic (abasic, AP) site is one of the most abundant DNA lesions. Previous studies by others demonstrated that human AlkB homologue 1 (ALKBH1) catalyzes the DNA strand incision at an AP site, resulting in suicidal cross-linking of the enzyme to the 3′-DNA end. Prior site-directed mutagenesis experiments had reported that Cys129 of ALKBH1 is the predominant nucleophile that conjugates to the C3′ position of the incised AP site, 3′-phospho-α,β-unsaturated aldehyde (3′-PUA), to form a 3′-PUA-ALKBH1 cross-link. However, direct evidence to support this mechanism was lacking. The 3′-PUA-ALKBH1 cross-link is so far the only adduct that has been found to form via a Michael addition reaction between a protein and 3′-PUA. It is unclear whether and how this type of cross-link is repaired. In this study, we first demonstrated that the 3′-PUA-ALKBH1 cross-link is fairly stable under physiological temperature and pH as only ~10% of the adduct decomposed after a 3-day incubation. Using a gel-based assay with an aldehyde-reacting probe, we demonstrated that the 3′-PUA-ALKBH1 cross-link has a free aldehyde group that is in line with the Michael addition mechanism. Moreover, we found that the 3′-PUA-ALKBH1 cross-link can be excised by human tyrosyl-DNA phosphodiesterase 1 (TDP1) and the removal efficiency is significantly enhanced if the adduct is pre-digested by trypsin. Notably, we employed TDP1 as a molecular tool to homogeneously release the cross-linked peptides from DNA to facilitate liquid chromatography tandem mass spectrometry analysis, and demonstrated that Cys129 and Cys371 of ALKBH1 cross-link to 3′-PUA. • The 3′-PUA-ALKBH1 cross-link is unstable but long-lived. • ALKBH1 cross-links to 3′-PUA through a Michael addition reaction. • Human TDP1 excises the 3′-PUA-ALKBH1 cross-link. • Proteolysis facilitates the 3′-PUA-ALKBH1 cross-link repair by TDP1. • A LC-MS/MS approach identified Cys129 and 371 of ALKBH1 that cross-link to 3′-PUA. [ABSTRACT FROM AUTHOR]

Base excision repair is the major pathway for the repair of oxidatively-induced DNA damage, with DNA glycosylases removing modified bases in the first step. Human NTHL1 is specific for excision of several pyrimidine- and purine-derived lesions from DNA, with loss of function NTHL1 showing a predisposition to carcinogenesis. A rare single nucleotide polymorphism of the Nthl1 gene leading to the substitution of Asp239 with Tyr within the active site, occurs within global populations. In this work, we overexpressed and purified the variant NTHL1-Asp239Tyr (NTHL1-D239Y) and determined the substrate specificity of this variant relative to wild-type NTHL1 using gas chromatography-tandem mass spectrometry with isotope-dilution, and oxidatively-damaged genomic DNA containing multiple pyrimidine- and purine-derived lesions. Wild-type NTHL1 excised seven DNA base lesions with different efficiencies, whereas NTHL1-D239Y exhibited no glycosylase activity for any of these lesions. We also measured the activities of human glycosylases OGG1 and NEIL1, and E. coli glycosylases Nth and Fpg under identical experimental conditions. Different substrate specificities among these DNA glycosylases were observed. When mixed with NTHL1-D239Y, the activity of NTHL1 was not reduced, indicating no substrate binding competition. These results and the inactivity of the variant D239Y toward the major oxidatively-induced DNA lesions points to the importance of the understanding of this variant's role in carcinogenesis and the potential of individual susceptibility to cancer in individuals carrying this variant. [ABSTRACT FROM AUTHOR]

DNA polymerases β (Pol β) and λ (Pol λ) belong to one structural family (X family) and possess the same enzymatic activities. Nonetheless, these enzymes have differences in their catalytic efficiency and specificity. We have previously reported that these enzymes can bypass bulky benzo[ a ]pyrene–DNA adducts via translesion synthesis during gap-filling reactions, although efficiency and specificity are dependent on the reaction conditions and adduct conformation. In the present study, we analyzed structural features of Pols β and λ complexed with a gapped DNA duplex containing either cis - or trans -benzo[ a ]pyrene-diol epoxide- N 2-dG (BP-dG) using molecular dynamics simulations. It was found that the most pronounced structural difference lies in the positioning of the trans -BP-dG residue relative to secondary structures of the protein; this dissimilarity may explain the differences between Pols β and λ in gap-filling/translesion synthesis. In the case of Pol β, trans -BP-dG turned out to be positioned parallel to the α-helix and β-sheet. In the Pol λ complex, trans -BP-dG is perpendicular to the α-helix. This difference persisted throughout the molecular dynamics trajectory. Selectivity for the BP-dG isomers remained after a deletion of noncatalytic domains of Pol λ. Modeling of Pol λ or β complexes with cis -BP-dG–containing DNA in the presence of Mn2+ either at both metal-binding sites or at the catalytic site only revealed that for both enzymes, the model of the complex containing both Mg2+ and Mn2+ is stabler than that containing two Mn2+ ions. This observation may reflect a shared property of these enzymes: the preference for Mn2+ in terms of catalysis and for Mg2+ regarding triphosphate coordination during the translesion reaction. • The positioning of trans -BP-dG differs in the DNA complexes with Pol λ and Pol β. • Pol λ N-terminal domains have no effect on the selectivity towards BP-dG conformers. • A model of the Pol λ complex with Mg2+ and Mn2+ is more stable than that with Mn2+. • In the course of simulation, one Mn2+ ion leaves Pol β complex with the cis -BP-dG-DNA. [ABSTRACT FROM AUTHOR]

3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:C T:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ . To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53 -mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:C T:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers’ lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity. [ABSTRACT FROM AUTHOR]

The cellular response to alkylation damage is complex, involving multiple DNA repair pathways and checkpoint proteins, depending on the DNA lesion, the cell type, and the cellular proliferation state. The repair of and response to O-alkylation damage, primarily O6-methylguaine DNA adducts (O6-mG), is the purview of O6-methylguanine-DNA methyltransferase (MGMT). Alternatively, this lesion, if left un-repaired, induces replication-dependent formation of the O6-mG:T mis-pair and recognition of this mis-pair by the post-replication mismatch DNA repair pathway (MMR). Two models have been suggested to account for MMR and O6-mG DNA lesion dependent formation of DNA double-strand breaks (DSBs) and the resulting cytotoxicity – futile cycling and direct DNA damage signaling. While there have been hints at crosstalk between the MMR and base excision repair (BER) pathways, clear mechanistic evidence for such pathway coordination in the formation of DSBs has remained elusive. However, using a novel protein capture approach, Fuchs and colleagues have demonstrated that DSBs result from an encounter between MMR-induced gaps initiated at alkylation induced O6-mG:C sites and BER-induced nicks at nearby N-alkylation adducts in the opposite strand. The accidental encounter between these two repair events is causal in the formation of DSBs and the resulting cellular response, documenting a third model to account for O6-mG induced cell death in non-replicating cells. This graphical review highlights the details of this Repair Accident model, as compared to current models, and we discuss potential strategies to improve clinical use of alkylating agents such as temozolomide, that can be inferred from the Repair Accident model. [ABSTRACT FROM AUTHOR]

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1, N 6 -ethenoadenine (ɛA), 3, N 4 -ethenocytosine (ɛC) and 1, N 2 -ethenoguanine (1, N 2 -ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis ) and SC-2B ( Streptomyces coelicolor ) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A ( Rhizobium etli ), SA-2B ( Streptomyces avermitilis ), and XC-2B ( Xanthomonas campestris ) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB ( Escherichia coli ), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1, N 2 -ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1, N 2 -ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. [ABSTRACT FROM AUTHOR]

Oxidative-stress-driven lipid peroxidation (LPO) is involved in the pathogenesis of several human diseases, including cancer. LPO products react with cellular proteins changing their properties, and with DNA bases to form mutagenic etheno-DNA adducts, removed from DNA mainly by the base excision repair (BER) pathway. One of the major reactive aldehydes generated by LPO is 4-hydroxy-2-nonenal (HNE). We investigated the effect of HNE on BER enzymes in human cells and in vitro . K21 cells pretreated with physiological HNE concentrations were more sensitive to oxidative and alkylating agents, H 2 O 2 and MMS, than were untreated cells. Detailed examination of the effects of HNE on particular stages of BER in K21 cells revealed that HNE decreases the rate of excision of 1, N 6 -ethenoadenine (ɛA) and 3, N 4 -ethenocytosine (ɛC), but not of 8-oxoguanine. Simultaneously HNE increased the rate of AP-site incision and blocked the re-ligation step after the gap-filling by DNA polymerases. This suggested that HNE increases the number of unrepaired single-strand breaks (SSBs) in cells treated with oxidizing or methylating agents. Indeed, preincubation of cells with HNE and their subsequent treatment with H 2 O 2 or MMS increased the number of nuclear poly(ADP-ribose) foci, known to appear in cells in response to SSBs. However, when purified BER enzymes were exposed to HNE, only ANPG and TDG glycosylases excising ɛA and ɛC from DNA were inhibited, and only at high HNE concentrations. APE1 endonuclease and 8-oxoG-DNA glycosylase 1 (OGG1) were not inhibited. These results indicate that LPO products exert their promutagenic action not only by forming DNA adducts, but in part also by compromising the BER pathway. [ABSTRACT FROM AUTHOR]

The accumulation of DNA damage is thought to contribute to the physiological decay associated with the aging process. Here, we report the results of a large-scale study examining longevity in various mouse models defective in the repair of DNA alkylation damage, or defective in the DNA damage response. We find that the repair of spontaneous DNA damage by alkyladenine DNA glycosylase (Aag/Mpg)-initiated base excision repair and O6-methylguanine DNA methyltransferase (Mgmt)-mediated direct reversal contributes to maximum life span in the laboratory mouse. We also uncovered important genetic interactions between Aag, which excises a wide variety of damaged DNA bases, and the DNA damage sensor and signaling protein, Atm. We show that Atm plays a role in mediating survival in the face of both spontaneous and induced DNA damage, and that Aag deficiency not only promotes overall survival, but also alters the tumor spectrum in Atm-/- mice. Further, the reversal of spontaneous alkylation damage by Mgmt interacts with the DNA mismatch repair pathway to modulate survival and tumor spectrum. Since these aging studies were performed without treatment with DNA damaging agents, our results indicate that the DNA damage that is generated endogenously accumulates with age, and that DNA alkylation repair proteins play a role in influencing longevity. [ABSTRACT FROM AUTHOR]

Abstract: The first eukaryotic NER factor that recognizes NER substrates is the heterodimeric XPC-RAD23B protein. The currently accepted hypothesis is that this protein recognizes the distortions/destabilization caused by DNA lesions rather than the lesions themselves. The resulting XPC-RAD23B–DNA complexes serve as scaffolds for the recruitment of subsequent NER factors that lead to the excision of the oligonucleotide sequences containing the lesions. Based on several well-known examples of DNA lesions like the UV radiation-induced CPD and 6–4 photodimers, as well as cisplatin-derived intrastrand cross-linked lesions, it is generally believed that the differences in excision activities in human cell extracts is correlated with the binding affinities of XPC-RAD23B to these DNA lesions. However, using electrophoretic mobility shift assays, we have found that XPC-RAD23B binding affinities of certain bulky lesions derived from metabolically activated polycyclic aromatic hydrocarbon compounds such as benzo[a]pyrene and dibenzo[a,l]pyrene, are not directly, or necessarily correlated with NER excision activities observed in cell-free extracts. These findings point to features of XPC-RAD23B–bulky DNA adduct complexes that may involve the formation of NER-productive or unproductive forms of binding that depend on the structural and stereochemical properties of the DNA adducts studied. The pronounced differences in NER cleavage efficiencies observed in cell-free extracts may be due to differences in the successful recruitment of subsequent NER factors by the XPC-RAD23B–DNA adduct complexes, and/or in the verification step. These phenomena appear to depend on the structural and conformational properties of the class of bulky DNA adducts studied. [Copyright &y& Elsevier]

Abstract: The concept of the Exposome is a compilation of diseases and one's lifetime exposure to chemicals, whether the exposure comes from environmental, dietary, or occupational exposures; or endogenous chemicals that are formed from normal metabolism, inflammation, oxidative stress, lipid peroxidation, infections, and other natural metabolic processes such as alteration of the gut microbiome. In this review, we have focused on the endogenous exposome, the DNA damage that arises from the production of endogenous electrophilic molecules in our cells. It provides quantitative data on endogenous DNA damage and its relationship to mutagenesis, with emphasis on when exogenous chemical exposures that produce identical DNA adducts to those arising from normal metabolism cause significant increases in total identical DNA adducts. We have utilized stable isotope labeled chemical exposures of animals and cells, so that accurate relationships between endogenous and exogenous exposures can be determined. Advances in mass spectrometry have vastly increased both the sensitivity and accuracy of such studies. Furthermore, we have clear evidence of which sources of exposure drive low dose biology that results in mutations and disease. These data provide much needed information to impact quantitative risk assessments, in the hope of moving towards the use of science, rather than default assumptions. [Copyright &y& Elsevier]

Distinct cellular DNA damage repair pathways maintain the structural integrity of DNA and protect it from the mutagenic effects of genotoxic exposures and processes. The occurrence of O 6-carboxymethylguanine (O 6-CMG) has been linked to meat consumption and hypothesized to contribute to the development of colorectal cancer. However, the cellular fate of O 6-CMG is poorly characterized and there is contradictory data in the literature as to how repair pathways may protect cells from O 6-CMG mutagenicity. To better address how cells detect and remove O 6-CMG, we evaluated the role of two DNA repair pathways in counteracting the accumulation and toxic effects of O 6-CMG. We found that cells deficient in either the direct repair protein O 6-methylguanine-DNA methyltransferase (MGMT), or key components of the nucleotide excision repair (NER) pathway, accumulate higher levels O 6-CMG DNA adducts than wild type cells. Furthermore, repair-deficient cells were more sensitive to carboxymethylating agents and displayed an increased mutation rate. These findings suggest that a combination of direct repair and NER circumvent the effects O 6-CMG DNA damage. [ABSTRACT FROM AUTHOR]

Highlights: [•] We developed a system for tracing DNA adducts in targeted mutagenesis (TATAM). [•] TATAM introduces synthetic DNA adducts into specific regions of the human genome. [•] We investigated the consequent mutations of synthetic 8-oxoG by TATAM. [•] The 8-oxoG is processed in the same manner as that generated by oxidative damage. [•] TATAM could be used in cells with altered expression of bypass or repair proteins. [Copyright &y& Elsevier]

Highlights: [•] Human cell lines that express a variant form of Pol κ (F171A) were established. [•] The cells exhibited lower mutation frequency toward benzo[a]pyrene DNA adducts. [•] Phenylalanine 171 is a molecular brake for the translesion DNA synthesis by Pol κ. [•] Pol κ is not well tuned to bypass benzo[a]pyrene DNA adducts. [•] The cognate substances for Pol κ may be lesions induced by endogenous mutagens. [Copyright &y& Elsevier]

Highlights: [•] Escherichia coli DinB and human pol κ discriminate against major groove modified DNA bases. [•] Activity is lowest with DNA templates containing ɛdA compared to N 6-furfuryl-dA. [•] Dpo4 is more accommodating of these modified bases than DinB or pol κ. [•] DinB R35A mutation eliminates discrimination against N 6-furfuryl-dA. [ABSTRACT FROM AUTHOR]

• NEIL2 is involved in the preferential repair of oxidized DNA base lesions from the actively transcribed genes in mammals. • BER enzymes function as a preformed complex with multiple other proteins forming a "BERosome". • NEIL2 has anti-inflammatory function in addition to its role in TC-BER. • TC-BER can be reconstituted in vitro using purified NEIL2, PNKP, Pol β , Ligase IIIα, RNAPII and essential transcription factors. • TC-BER deficiency can be linked to various human pathological conditions. Oxidized bases in the genome has been implicated in various human pathologies, including cancer, aging and neurological diseases. Their repair is initiated with excision by DNA glycosylases (DGs) in the base excision repair (BER) pathway. Among the five oxidized base-specific human DGs, OGG1 and NTH1 preferentially excise oxidized purines and pyrimidines, respectively, while NEILs remove both oxidized purines and pyrimidines. However, little is known about why cells possess multiple DGs with overlapping substrate specificities. Studies of the past decades revealed that some DGs are involved in repair of oxidized DNA base lesions in the actively transcribed regions. Preferential removal of lesions from the transcribed strands of active genes, called transcription-coupled repair (TCR), was discovered as a distinct sub-pathway of nucleotide excision repair; however, such repair of oxidized DNA bases had not been established until our recent demonstration of NEIL2's role in TC-BER of the nuclear genome. We have shown that NEIL2 forms a distinct transcriptionally active, repair proficient complex. More importantly, we for the first time reconstituted TC-BER using purified components. These studies are important for characterizing critical requirement for the process. However, because NEIL2 cannot remove all types of oxidized bases, it is unlikely to be the only DNA glycosylase involved in TC-BER. Hence, we postulate TC-BER process to be universally involved in maintaining the functional integrity of active genes, especially in post-mitotic, non-growing cells. We further postulate that abnormal bases (e.g. , uracil), and alkylated and other small DNA base adducts are also repaired via TC-BER. In this review, we have provided an overview of the various aspects of TC-BER in mammalian cells with the hope of generating significant interest of many researchers in the field. Further studies aimed at better understanding the mechanistic aspects of TC-BER could help elucidate the linkage of TC-BER deficiency to various human pathologies. [ABSTRACT FROM AUTHOR]

Abstract: Rad9, an evolutionarily conserved checkpoint gene with multiple functions for preserving genomic integrity, has been shown to play important roles in homologous recombination repair, base excision repair and mismatch repair. However, whether Rad9 has an impact on nucleotide excision repair remains unknown. Here we demonstrated that Rad9 was involved in nucleotide excision repair and loss of Rad9 led to defective removal of the UV-derived photoproduct 6-4PP (6,4 pyrimidine-pyrimidone) and the BPDE (anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide)–DNA adducts in mammalian cells. We also demonstrated that Rad9 could co-localize with XPC in response to local UV irradiation. However, our data showed that Rad9 was not required for the photoproducts recognition step of nucleotide excision repair. Further investigation revealed that reduction of Rad9 reduced the UV-induced transcription of the genes of the nucleotide excision repair factors DDB2, XPC, DDB1 and XPB and DDB2 protein levels in human cells. Interestingly, knockdown of one subunit of DNA damage recognition complex, hHR23B impaired Rad9-loading onto UV-damaged chromatin. Based on these results, we suggest that Rad9 plays an important role in nucleotide excision repair through mechanisms including maintaining DDB2 protein level in human cells. [Copyright &y& Elsevier]

Abstract: Neurodevelopmental defects are observed in the hereditary disorder Cockayne syndrome (CS). The gene most frequently mutated in CS, Cockayne Syndrome B (CSB), is required for the repair of bulky DNA adducts in transcribed genes during transcription-coupled nucleotide excision repair. CSB also plays a role in chromatin remodeling and mitochondrial function. The role of CSB in neural development is poorly understood. Here we report that the abundance of neural progenitors is normal in Csb −/− mice and the frequency of apoptotic cells in the neurogenic niche of the adult subependymal zone is similar in Csb −/− and wild type mice. Both embryonic and adult Csb −/− neural precursors exhibited defective self-renewal in the neurosphere assay. In Csb −/− neural precursors, self-renewal progressively decreased in serially passaged neurospheres. The data also indicate that Csb and the nucleotide excision repair protein Xpa preserve embryonic neural stem cell self-renewal after UV DNA damage. Although Csb −/− neural precursors do not exhibit altered neuronal lineage commitment after low-dose UV (1J/m2) in vitro, neurons differentiated in vitro from Csb −/− neural precursors that had been irradiated with 1J/m2 UV exhibited defective neurite outgrowth. These findings identify a function for Csb in neural precursors. [Copyright &y& Elsevier]

Abstract: The combined action of oxidative stress and genotoxic polycyclic aromatic hydrocarbons derivatives can lead to cluster-type DNA damage that includes both a modified nucleotide and a bulky lesion. As an example, we investigated the possibility of repair of an AP site located opposite a minor groove–positioned (+)-trans-BPDE-dG or a base-displaced intercalated (+)-cis-BPDE-dG adduct (BP lesion) by a BER system. Oligonucleotides with single uracil residues in certain positions were annealed with complementary oligonucleotides bearing either a cis- or trans-BP adduct. The resulting DNA duplexes contained dU either directly opposite the modified dG or shifted to adjacent 5′ (−1) or 3′ (+1) positions. Digestion with uracil DNA glycosylase was utilized to generate AP sites which were then hydrolyzed by APE1, and the resulting gaps were processed by DNA polymerase β (Polβ) or λ (Polλ). The AP sites in position −1 can be repaired effectively using APE1 and Polβ or Polλ. The AP sites opposite the BP lesions can be repaired using Polλ in the case of cis- but not the trans-isomeric adduct. The AP sites in position +1 are the most difficult to repair. In the case of the AP site located in position +1, the activity of Polλ does not depend on the stereoisomeric properties of the BP lesions and dCTP is the preferred inserted substrate in both cases. The capability of Polλ to introduce the correct dNTP opposite the cis-BP-dG adduct in gap filling reactions suggests that this polymerase may play a specialized role in the process of repair of these kinds of lesions. [Copyright &y& Elsevier]

Abstract: To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2′-deoxycytidine-5′-triphosphate, and [32P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3′-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase ɛ and Ddc1 checkpoint protein. Labeling of DNA polymerase ɛ catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA–protein adducts. [Copyright &y& Elsevier]

Abstract: Polycyclic aromatic hydrocarbons (PAH) are an important class of environmental contaminants many of which require metabolic activation to DNA-reactive bay or fjord region diolepoxides (DE) in order to exert their mutagenic and carcinogenic effects. In this study, the mutagenicity of the bay region diolepoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (±)-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenzo[a,h]anthracene (DBADE) and the fjord region diolepoxides (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]-pyrene (DBPDE) and (±)-anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene (BPhDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. The 32P-postlabelling assay was applied to analyze DNA adduct levels and the Hprt gene mutation assay for monitoring mutations. Previously, we found that the mutagenicity per adduct was four times higher for DBPDE compared to BPDE in NER proficient cells. In these same cells, the mutagenicity of DBADE and BPhDE adducts was now found to be significantly lower compared to that of BPDE. In NER deficient cells the highest mutagenicity per adduct was found for BPDE and there was a tenfold and fivefold difference when comparing the BPDE data with the DBADE and BPhDE data, respectively. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the Hprt gene. Since NER turned out to be an important pathway for the yield of mutations, we further analyzed the role of transcription coupled NER versus global genome NER. However, our data demonstrate that neither of these pathways seems to be the sole factor determining the mutation frequency of the four PAH–DE and that the differences in the repair efficiency of these compounds could not be related to the presence of a bay or fjord region in the parent PAH. [Copyright &y& Elsevier]

Abstract: Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism that removes a large variety of structurally distinct lesions in cellular DNA. While the proteins involved are completely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryotic and eukaryotic NER damage-recognition factors have common structural features of β-hairpin intrusion between the two DNA strands at the site of the lesion. In the present study, we explored the hypothesis that this common β-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the two systems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determine their relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with a set of stereoisomeric DNA lesions derived from metabolites of benzo[a]pyrene and equine estrogens in different sequence contexts, utilizing 21 samples. We found a general qualitative trend toward similar relative NER incision efficiencies for ∼65% of these substrates; the other cases deviate mostly by ∼30% or less from a perfect correlation, although several more distant outliers are also evident. This resemblance is consistent with the hypothesis that lesion recognition through β-hairpin insertion, a common feature of the two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases. In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining the same UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggest that, in addition to recognition involving the initial modified duplexes, downstream events involving UvrC can also play a role in distinguishing and processing different lesions in prokaryotic NER. [Copyright &y& Elsevier]

Abstract: Telomeres are nucleoprotein complexes at the ends of linear chromosomes in eukaryotes, and are essential in preventing chromosome termini from being recognized as broken DNA ends. Telomere shortening has been linked to cellular senescence and human aging, with oxidative stress as a major contributing factor. 7,8-Dihydro-8-oxogaunine (8-oxodG) is one of the most abundant oxidative guanine lesions, and 8-oxoguanine DNA glycosylase (OGG1) is involved in its removal. In this study, we examined if telomeric DNA is particularly susceptible to oxidative base damage and if telomere-specific factors affect the incision of oxidized guanines by OGG1. We demonstrated that telomeric TTAGGG repeats were more prone to oxidative base damage and repaired less efficiently than non-telomeric TG repeats in vivo. We also showed that the 8-oxodG-incision activity of OGG1 is similar in telomeric and non-telomeric double-stranded substrates. In addition, telomere repeat binding factors TRF1 and TRF2 do not impair OGG1 incision activity. Yet, 8-oxodG in some telomere structures (e.g., fork-opening, 3′-overhang, and D-loop) were less effectively excised by OGG1, depending upon its position in these substrates. Collectively, our data indicate that the sequence context of telomere repeats and certain telomere configurations may contribute to telomere vulnerability to oxidative DNA damage processing. [ABSTRACT FROM AUTHOR]

Abstract: Polycyclic aromatic hydrocarbons (PAHs) are carcinogens with varying potencies. These compounds are metabolized to diol epoxides that react to form DNA adducts. Nucleotide excision repair is a critical cellular defense against these bulky DNA adducts which, if not repaired, can lead to mutations and the initiation of cancer. The structural features of the PAH-adducts play a role in differential repair of these adducts by the global genomic repair subpathway of nucleotide excision repair. DNA adducts derived from the PAHs containing bay-regions are repaired more rapidly than adducts derived from PAHs containing fjord-regions. We have employed the host cell reactivation assay to examine the rate of repair of these adducts in an actively transcribing gene. The pGL3 plasmid containing a luciferase gene was damaged with diol epoxides of benzo[a]pyrene (B[a]P-DE), dibenzo[a,l]pyrene (DB[a,l]P-DE), benzo[g]chrysene (B[g]Ch-DE), and benzo[c]phenanthrene (B[c]Ph-DE). The plasmids were transfected into B-lymphocytes with normal repair capacity as well as lymphocytes derived from patients with the XP-A, XP-C and CS-B syndromes. We found that XPA cells were able to transcribe slowly past B[g]Ch-adducts but not the other PAHs. Using the amount of luciferase produced as a measure of DNA repair, we found that the relative rates of repair in the actively transcribing luciferase gene was B[a]P-DE>DB[a,l]P-DE, B[g]Ch-DE, >B[c]Ph-DE in repair proficient and XP-C cells. These results indicate that the abilities to transcribe past and to repair the PAH adducts are dependent on different structural features of the DNA adducts. [ABSTRACT FROM AUTHOR]

Abstract: Mutations induced by polycyclic aromatic hydrocarbons (PAH) are expected to be produced when error-prone DNA replication occurs across unrepaired DNA lesions formed by reactive PAH metabolites such as diol epoxides. The mutagenicity of the two PAH-diol epoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. We applied the 32P-postlabelling assay to analyze adduct levels and the hprt gene mutation assay for monitoring mutations. It was found that the mutagenicity per target dose was 4 times higher for DBPDE compared to BPDE in NER proficient cells while in NER deficient cells, the mutagenicity per target dose was 1.4 times higher for BPDE. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the hprt gene. The results suggest that NER of BPDE lesions are 5 times more efficient than for DBPDE lesions, in NER proficient cells. However, DBPDE adducts block replication more efficiently and also induce 6 times more recombination events in the hprt gene than adducts of BPDE, suggesting that DBPDE adducts are, to a larger extent, bypassed by homologous recombination. The results obtained here indicate that the mutagenicity of PAH is influenced not only by NER, but also by replication bypass fidelity. This has been postulated earlier based on results using in vitro enzyme assays, but is now also being recognized in terms of forward mutations in intact mammalian cells. [Copyright &y& Elsevier]

Abstract: Benzo[a]pyrene exerts its mutagenic effects via induction of benzo[a]pyrene-diol-epoxide (BPDE)–DNA adducts. Such helix-distorting adducts are not always successfully repaired prior to DNA replication, which may result in a blocked replication fork. To alleviate this stall, cells utilize DNA damage tolerance systems involving either error-free damage avoidance or error-prone translesion synthesis. Studies in yeast suggest the modification of PCNA by lysine 63-linked poly-ubiquitin (K63-polyUb) chains as a key mediator of the error-free damage avoidance pathway. Recently, we extended this observation to human cells, showing the occurrence of poly-ubiquitination of PCNA in UV-irradiated human cells. In the present study, we hypothesized that disrupting the formation of K63-polyUb chains inhibits damage avoidance and favors error-prone repair involving low-fidelity polymerases (e.g. POLη), causing increased BPDE-induced mutagenicity. To test this hypothesis, we generated A549 cells expressing either a mutant ubiquitin (K63R-Ub) which blocks further ubiquitination through K63, or the wild type ubiquitin (WT-Ub). We show that PCNA is poly-ubiquitinated in these cells upon BPDE-exposure and that disruption of K63-polyUb chain formation has no effect on BPDE-induced toxicity. In contrast, significantly higher frequencies of BPDE-induced HPRT mutations were observed in K63R-Ub expressing cells, of which the majority (74%) was G→T transversion. BPDE treatment caused an enhanced recruitment of POLη to the replication machinery of the K63R-Ub expressing cells, where it co-localized with PCNA. Suppression of POLη expression by using siRNA resulted in a 50% reduction of BPDE-induced mutations in the K63R cells. In conclusion, we demonstrated that formation of K63-polyUb chains protects BPDE-exposed human cells against translesion synthesis-mediated mutagenesis. These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by preventing mutagenesis and thus contributing to genomic stability. [Copyright &y& Elsevier]

Abstract: Benzene is a human leukemia carcinogen, resulting from its cellular metabolism. A major benzene metabolite is p-benzoquinone (pBQ), which can damage DNA by forming the exocyclic base adducts pBQ-dC, pBQ-dA, and pBQ-dG in vitro. To gain insights into the role of pBQ in benzene genotoxicity, we examined in vitro translesion synthesis and in vivo mutagenesis of these pBQ adducts. Purified REV1 and Polκ were essentially incapable of translesion synthesis in response to the pBQ adducts. Opposite pBQ-dA and pBQ-dC, purified human Polι was capable of error-prone nucleotide insertion, but was unable to perform extension synthesis. Error-prone translesion synthesis was observed with Polη. However, DNA synthesis largely stopped opposite the lesion. Consistent with in vitro results, replication of site-specifically damaged plasmids was strongly inhibited by pBQ adducts in yeast cells, which depended on both Polζ and Polη. In wild-type cells, the majority of translesion products were deletions at the site of damage, accounting for 91%, 90%, and 76% for pBQ-dA, pBQ-dG, and pBQ-dC, respectively. These results show that the pBQ-dC, pBQ-dA, and pBQ-dG adducts are strong blocking lesions, and are highly mutagenic by predominantly inducing deletion mutations. These results are consistent with the lesion structures predicted by molecular dynamics simulation. Our results led to the following model. Translesion synthesis normally occurs by directly copying the lesion site through base insertion and extension synthesis. When the lesion becomes incompatible in accommodating a base opposite the lesion in DNA, translesion synthesis occurs by a less efficient lesion loop-out mechanism, resulting in avoiding copying the damaged base and leading to deletion. [Copyright &y& Elsevier]

The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF–nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells. [Copyright &y& Elsevier]

In cells, the major benzo[a]pyrene DNA adduct is the highly mutagenic (+)-trans-anti-BPDE-N2-dG. In eukaryotes, little is known about lesion bypass of this DNA adduct during replication. Here, we show that purified human Polκ can effectively bypass a template (+)-trans-anti-BPDE-N2-dG adduct in an error-free manner. Kinetic parameters indicate that Polκ bypass of the (−)-trans-anti-BPDE-N2-dG adduct was ∼41-fold more efficient compared to the (+)-trans-anti-BPDE-N2-dG adduct. Furthermore, we have found another activity of human Polκ in response to the (+)- and (−)-trans-anti-BPDE-N2-dG adducts: extension synthesis from mispaired primer 3′ ends opposite the lesion. In contrast, the two adducts strongly blocked DNA synthesis by the purified human Polβ and the purified catalytic subunits of yeast Polα, Polδ, and Polϵ right before the lesion. Extension by human Polκ from the primer 3′ G opposite the (+)- and (−)-trans-anti-BPDE-N2-dG adducts was mediated by a −1 deletion mechanism, probably resulting from re-aligning the primer G to pair with the next template C by Polκ prior to DNA synthesis. Thus, sequence contexts 5′ to the lesion strongly affect the fidelity and mechanism of the Polκ-catalyzed extension synthesis. These results support a dual-function model of human Polκ in bypass of BPDE DNA adducts: it may function both as an error-free bypass polymerase alone and an extension synthesis polymerase in combination with another polymerase. [ABSTRACT FROM AUTHOR]

Graph It is with great sadness we announce that Dr. Takashi Sugimura passed away on September 6 SP th sp , 2020 from heart failure at the age of 94. By seeing that many carcinogens cause DNA adducts and the generated mutations in the genome are the driving force of multi-step carcinogenesis, Sugimura became deeply interested in DNA repair mechanisms as well. In 1973, Sugimura and Hayaishi organized a meeting on poly(ADP-ribose) in Bethesda, Washington then in 1974, in Tomakomai, Japan and in 1982 in Tokyo. Followed by the finding of PARP and its inhibitors (PARPi), Sugimura showed that a PARPi benzamide potentiated the anti-tumor activity of bleomycin in Ehrlich's ascites cancer model of mice, in 1983 [[11]]. [Extracted from the article]

• Elucidates a mechanism of cell killing by 5-aza-dC. • Presents a new method, Adduct-Seq, for mapping protein-DNA adducts genomewide. • Identifies targets and consequences of PARP1-DNA adduction in living cells. • Challenges the view of 5-aza-dC (decitabine) as an "epigenetic" drug. The nucleoside analog 5-aza-2′-deoxycytidine (5-aza-dC) is used to treat some hematopoietic malignancies. The mechanism of cell killing depends upon DNMT1, but is otherwise not clearly defined. Here we show that PARP1 forms covalent DNA adducts in human lymphoblast or fibroblasts treated with 5-aza-dC. Some adducts recovered from 5-aza-dC-treated cells have undergone cleavage by apoptotic caspases 3/7. Mapping of PARP1-DNA adducts, by a new method, "Adduct-Seq", demonstrates adduct enrichment at CpG-dense genomic locations that are targets of maintenance methylation by DNMT1. Covalent protein-DNA adducts can arrest replication and induce apoptosis, and these results raise the possibility that induction of PARP1-DNA adducts may contribute to cell killing in response to treatment with 5-aza-dC. [ABSTRACT FROM AUTHOR]

• π–π interactions play key roles in positioning AlkB and ALKBH2 substrates for repair. • π–π interactions are disrupted when AlkB or ALKBH2 bind N2,3-εG. • Calculations predict small oxidation barriers for optimally placed substrates. • A large oxidation barrier is predicted for N2,3-εG repair. • The large barrier explains why N2,3-εG escapes AlkB-mediated repair. E. coli AlkB and human ALKBH2 belong to the AlkB family enzymes, which contain several α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases that repair alkylated DNA. Specifically, the AlkB enzymes catalyze decarboxylation of α-KG to generate a high-valent Fe(IV)-oxo species that oxidizes alkyl groups on DNA adducts. AlkB and ALKBH2 have been reported to differentially repair select etheno adducts, with preferences for 1, N 6-ethenoadenine (1,N6-εA) and 3, N 4-ethenocytosine (3,N4-εC) over 1, N 2-ethenoguanine (1,N2-εG). However, N 2,3-ethenoguanine (N2,3-εG), the most common etheno adduct, is not repaired by the AlkB enzymes. Unfortunately, a structural understanding of the differential activity of E. coli AlkB and human ALKBH2 is lacking due to challenges acquiring atomistic details for a range of substrates using experiments. This study uses both molecular dynamics (MD) simulations and ONIOM(QM:MM) calculations to determine how the active site changes upon binding each etheno adduct and characterizes the corresponding catalytic impacts. Our data reveal that the preferred etheno substrates (1,N6-εA and 3,N4-εC) form favorable interactions with catalytic residues that situate the lesion near the Fe(IV)-oxo species and permit efficient oxidation. In contrast, although the damage remains correctly aligned with respect to the Fe(IV)-oxo moiety, repair of 1,N2-εG is mitigated by increased solvation of the active site and a larger distance between Fe(IV)-oxo and the aberrant carbons. Binding of non-substrate N2,3-εG in the active site disrupts key DNA–enzyme interactions, and positions the aberrant carbon atoms even further from the Fe(IV)-oxo species, leading to prohibitively high barriers for oxidative catalysis. Overall, our calculations provide the first structural insight required to rationalize the experimentally-reported substrate specificities of AlkB and ALKBH2 and thereby highlight the roles of several active site residues in the repair of etheno adducts that directly correlates with available experimental data. These proposed catalytic strategies can likely be generalized to other α-KG/Fe(II)-dependent dioxygenases that play similar critical biological roles, including epigenetic and post-translational regulation. [ABSTRACT FROM AUTHOR]

6-Nitrochrysene (6-NC) is a potent mutagen in bacteria and carcinogenic in animals. It is the most potent carcinogen ever tested in newborn mouse assay. DNA lesions resulting from 6-NC modification are likely to induce mutations if they are not removed by cellular defense pathways prior to DNA replication. Earlier studies showed that 6-NC-derived C8−2'-deoxyadenosine adduct, N -(dA-8-yl)-6-AC, is very slowly repaired in human cells. In this study, we have investigated replication of N -(dA-8-yl)-6-AC in human embryonic kidney (HEK 293T) cells and the roles of translesion synthesis (TLS) DNA polymerases in bypassing it. Replication of a plasmid containing a single site-specific N -(dA-8-yl)-6-AC adduct in HEK 293 T cells showed that human DNA polymerase (hPol) η and hPol κ played important roles in bypassing the adduct, since TLS efficiency was reduced to 26 % in the absence of these two polymerases compared to 83 % in polymerase-competent HEK 293T cells. The progeny from HEK 293T cells provided 12.7 % mutants predominantly containing A→T transversions. Mutation frequency (MF) was increased to 17.8 % in hPol η-deficient cells, whereas it was decreased to 3.3 % and 3.9 % when the adduct containing plasmid was replicated in hPol κ- and hPol ζ-deficient cells, respectively. The greatest reduction in MF by more than 90 % (to MF 1.2 %) was observed in hPol ζ-knockout cells in which hPol κ was knocked down. Taken together, these results suggest that hPol κ and hPol ζ are involved in the error-prone TLS of N -(dA-8-yl)-6-AC, while hPol η performs error-free bypass. [ABSTRACT FROM AUTHOR]

Proteins that act on DNA, or are in close proximity to it, can become inadvertently crosslinked to DNA and form highly toxic lesions, known as DNA-protein crosslinks (DPCs). DPCs are generated by different chemotherapeutics, environmental or endogenous sources of crosslinking agents, or by lesions on DNA that stall the catalytic cycle of certain DNA processing enzymes. These bulky adducts impair processes on DNA such as DNA replication or transcription, and therefore pose a serious threat to genome integrity. The large diversity of DPCs suggests that there is more than one canonical mechanism to repair them. Indeed, many different enzymes have been shown to act on DPCs by either processing the protein, the DNA or the crosslink itself. In addition, the cell cycle stage or cell type are likely to dictate pathway choice. In recent years, a detailed understanding of DPC repair during S phase has started to emerge. Here, we review the current knowledge on the mechanisms of replication-coupled DPC repair, and describe and also speculate on possible pathways that remove DPCs outside of S phase. Moreover, we highlight a recent paradigm shifting finding that indicates that DPCs are not always detrimental, but can also play a protective role, preserving the genome from more deleterious forms of DNA damage. [ABSTRACT FROM AUTHOR]

Keywords: PARP-1, poly(ADP-ribose) polymerase 1; DPC, DNA-protein crosslink; BER, base excision repair; AP site/abasic site, apurinic/apyrimidinic site EN PARP-1, poly(ADP-ribose) polymerase 1 DPC, DNA-protein crosslink BER, base excision repair AP site/abasic site, apurinic/apyrimidinic site N.PAG N.PAG 1 06/15/20 20200601 NES 200601 1 Introduction Cellular DNA is constantly damaged by endogenous and exogenous DNA damaging agents, including both environmental physical and chemical agents, such as UV light and ionizing radiation [[1]]. 4 PARP-1 DPC formation localizes to the Zn3BRCT region of PARP-1 To evaluate the underlying mechanism of DPC formation, we studied covalent bond formation between PARP-1 and AP site-containing DNA as a function of PARP-1 domain structure. 10 Repair of PARP-1 DPC in vitro In assessing PARP-1 DPC repair in vitro, we were unable to isolate sufficient PARP-1 DPC from mouse embryonic fibroblasts to test the hypothesis that the post-proteasome DPC is repaired by BER, in partnership with the protein degradation system. The migration positions of PARP-1 crosslinked to the full-length DNA strand (~127 kDa) and C3' incised DNA strand (~121 kDa) are indicated. The symbol ( ) represents PARP-1, AP represents the AP site in the DNA, XL-PARP-1 indicates crosslinked PARP-1, and 32P indicates the position of radiolabel on the DNA. [Extracted from the article]

• The "adductome" in human cells includes a limited set of proteins. • RADAR and buoyant density fractionation define highly concordant adductomes. • RADAR fractionation combined with MS can discover novel protein-nucleic acid adducts. • Proteins involved in mRNA processing are overrepresented in the adductome. • Proteins reactive with exogenous formaldehyde are enriched in the adductome. Proteins form adducts with nucleic acids in a variety of contexts, and these adducts may be cytotoxic if not repaired. Here we apply a proteomic approach to identification of proteins adducted to DNA or RNA in normally proliferating cells. This approach combines RADAR fractionation of proteins covalently bound to nucleic acids with quantitative mass spectrometry (MS). We demonstrate that "RADAR-MS" can quantify induction of TOP1- or TOP2-DNA adducts in cells treated with topotecan or etoposide, respectively, and also identify intermediates in physiological adduct repair. We validate RADAR-MS for discovery of previously unknown adducts by determining the repertoires of adducted proteins in two different normally proliferating human cell lines, CCRF-CEM T cells and GM639 fibroblasts. These repertoires are significantly similar with one another and exhibit robust correlations in their quantitative profiles (Spearman r = 0.52). A very similar repertoire is identified by the classical approach of CsCl buoyant density gradient centrifugation. We find that in normally proliferating human cells, the repertoire of adducted proteins — the "adductome" — is comprised of a limited number of proteins belonging to specific functional groups, and that it is greatly enriched for histones, HMG proteins and proteins involved in RNA splicing. Treatment with low concentrations of formaldehyde caused little change in the composition of the repertoire of adducted proteins, suggesting that reactive aldehydes generated by ongoing metabolic processes may contribute to protein adduction in normally proliferating cells. The identification of an endogenous adductome highlights the importance of adduct repair in maintaining genomic structure and the potential for deficiencies in adduct repair to contribute to cancer. [ABSTRACT FROM AUTHOR]

Cellular DNA is constantly damaged by endogenous and exogenous DNA damaging agents, including both environmental physical and chemical agents, such as UV light and ionizing radiation [[1]]. These results are consistent with strand incision proceeding through -elimination of the DNA from the C3' position of deoxyribose in the AP site; a labeled DNA-PARP-1 complex would not be observed since the radiolabel was on the 3'-end of the DNA strand (see Fig. 4 PARP-1 DPC formation localizes to the Zn3BRCT region of PARP-1 To evaluate the underlying mechanism of DPC formation, we studied covalent bond formation between PARP-1 and AP site-containing DNA as a function of PARP-1 domain structure. The migration positions of PARP-1 crosslinked to the full-length DNA strand (~127 kDa) and C3' incised DNA strand (~121 kDa) are indicated. The symbol () represents PARP-1, AP represents the AP site in the DNA, XL-PARP-1 indicates crosslinked PARP-1, and 32P indicates the position of radiolabel on the DNA. [Extracted from the article]

• Covalent DNA-protein crosslinks are highly-toxic endogenous DNA lesions. • The proteases Wss1 and SPRTN degrade the protein component of DNA-protein crosslinks. • Wss1 and SPRTN share an evolutionary origin but are not immediate orthologs. • Multiple partially-redundant proteolytic systems target DNA-protein crosslinks. Covalent DNA-protein crosslinks (DPCs) are highly toxic DNA adducts, which interfere with faithful DNA replication. The proteases Wss1 and SPRTN degrade DPCs and have emerged as crucially important DNA repair enzymes. Their protective role has been described in various model systems ranging from yeasts, plants, worms and flies to mice and humans. Loss of DPC proteases results in genome instability, cellular arrest, premature ageing and cancer predisposition. Here we discuss recent insights into the function and molecular mechanism of these enzymes. Furthermore, we present an in-depth phylogenetic analysis of the Wss1/SPRTN protease continuum. Remarkably flexible domain architectures and constantly changing protein-protein interaction motifs indicate ongoing evolutionary dynamics. Finally, we discuss recent data, which suggest that further partially-overlapping proteolytic systems targeting DPCs exist in eukaryotes. These new developments raise interesting questions regarding the division of labour between different DPC proteases and the mechanisms and principles of repair pathway choice. [ABSTRACT FROM AUTHOR]

It is to be noted that this publication originally appeared only On Line, in DNA Repair 11 (2012) 452e1-452e11. While Ray collaborated with me on replication intermediates and isolation of replication forks in normally replicating DNA [[24]], Dave focused upon UV-irradiated cells, with the rationale that we might catch DNA fragments in which replication forks had been blocked at a CPD; in addition, Ray carried out pioneering research on the characterization of DNA in the mitochondria and chloroplasts of Euglena gracilis [[25]]. The 8th annual meeting of the Biophysical Society in February 1964 began with a symposium, chaired by Henry Kaplan, on "Macromolecular aspects of radiation actions on cells", in which Bob Haynes spoke on "DNA as target for actions of X-rays, UV and radiomimetic chemicals", followed by talks from Setlow and Howard-Flanders on excision of pyrimidine dimers from DNA in UV-irradiated E. coli. In the following contributed paper session, Dan Ray, Dave Pettijohn and I each gave talks; on chloroplast DNA in Euglena, on physical properties of newly-synthesized DNA in UV-irradiated E. coli, and on characterization of the DNA at the replication point, respectively. [Extracted from the article]

• Experimentally observed NER efficiencies for lesions in nucleosomes are structurally interpreted with MD simulations. • Two bulky benzo[ a ]pyrene-derived lesions and a small cyclopurine cross-link have different impacts on nucleosome structure and dynamics. • Rotational and translational settings of the lesions play a key role in their impact on lesion and histone structures and interactions. • Lesion-governed differences in nucleosome structure and dynamics are proposed to signal the lesion access machinery. • The rate that access is achieved is hypothesized to contribute to NER efficiency. The packaging of DNA in nucleosomes presents a barrier for biological transactions including replication, transcription and repair. However, despite years of research, how the DNA is freed from the histone proteins and thereby allows the molecular machines to access the DNA remains poorly understood. We are interested in global genomic nucleotide excision repair (GG-NER). It is established that the histones are obstacles to this process, and DNA lesions are repaired less efficiently in nucleosomes than in free DNA. In the present study, we utilized molecular dynamics simulations to elucidate the nature of the distortions and dynamics imposed in the nucleosome by a set of three structually different lesions that vary in GG-NER efficiencies in free DNA, and in nucleosomes [Shafirovich, Geacintov, et. al, 2019]. Two of these are bulky lesions derived from metabolic activation of the environmental carcinogen benzo[ a ]pyrene, the 10 R (+)- cis-anti -B[ a ]P- N 2-dG and the stereoisomeric 10 S (+)- trans-anti -B[ a ]P- N 2-dG, which respectively adopt base-displaced/intercalated and minor groove-aligned conformations in DNA. The third is a non-bulky lesion, the 5′ R -8-cyclo-2′-deoxyguanosine cross-link, produced by reactive oxygen and nitrogen species; cyclopurine lesions are highly mutagenic. These adducts are placed near the dyad axis, and rotationally with the lesion-containing strand facing towards or away from the histones. While each lesion has distinct conformational characteristics that are retained in the nucleosome, a spectrum of structural and dynamic disturbances, from slight to substantial, are displayed that depend on the lesion's structure and position in the nucleosome. We hypothesize that these intrinsic structural and dynamic distinctions provide different signals to initiate the cascade of chromatin-opening processes, including acetylation and other post translational modifications, remodeling by ATP-dependent complexes and spontaneous unwrapping that regulate the rate of access to the lesion; this may translate ultimately into varying GG-NER efficiencies, including repair resistance when signals for access are too weak. [ABSTRACT FROM AUTHOR]

• HPV induces APOBEC3 expression in HNSC. • High APOBEC3 G expression correlates with better overall survival. • APOBEC3, Polβ and MSH6 knockdown results in cisplatin resistance. • Cisplatin resistance following A3 knockdown correlates with ICL repair. Human papillomavirus (HPV) is associated with the development of head and neck squamous cell carcinomas (HNSC). Cisplatin is used to treat HNSC and induces DNA adducts including interstrand crosslinks (ICLs). Previous reports have shown that HPV positive HNSC patients respond better to cisplatin therapy. Our previous reports highlight that loss of base excision repair (BER) and mismatch repair (MMR) results in cisplatin resistance. Of importance, uracil DNA glycosylase (UNG) is required to initiate the BER response to cisplatin treatment and maintain drug sensitivity. These previous results highlight that specific cytidine deaminases could play an important role in the cisplatin response by activating the BER pathway to mediate drug sensitivity. The APOBEC3 (A3) family of cytidine deaminases are enzymes that restrict HPV as part of the immune defense to viral infection. In this study, the Cancer Genome Atlas (TCGA) HNSC data were used to assess the association between the expression of the seven proteins in the A3 cytidine deaminase family, HPV-status and survival outcomes. Higher A3 G expression in HPV-positive tumors corresponds with better overall survival (OS) (HR 0.33, 95 % CI 0.11-0.93, p = 0.04). FaDu and Scc-25 HNSC cell lines were used to assess alterations in A3, BER and MMR expression in response to cisplatin. We demonstrate that A3, Polβ, and MSH6 knockdown in HNSC cells results in resistance to cisplatin and carboplatin as well as an increase in the rate of ICL removal in FaDu and Scc-25 HNSC cells. Our results suggest that A3s activate BER in HNSC, mediate repair of cisplatin ICLs and thereby, sensitize cells to cisplatin which likely contributes to the improved patient responses observed in HPV infected patients. [ABSTRACT FROM AUTHOR]

• This article characterizes a novel pathway for interstrand DNA cross-link (ICL) repair. • This ICL repair pathway involves the unhooking of the cross-link by a glycosylase enzyme NEIL3. • NEIL3 selectively unhooks a dA-AP ICL located at the junction duplex/single strand junction of a model replication fork. • The dA-AP ICL is selectively unhooked when the AP site is located on the leading strand template. Interstrand DNA-DNA cross-links (ICLs) are generated by endogenous processes, drugs, and environmental toxins. Understanding the cellular pathways by which various ICLs are repaired is critical to understanding their biological effects. Recent studies showed that replication-dependent repair of an ICL derived from the reaction of an abasic (AP) site with an adenine residue (dA) on the opposing strand of duplex DNA proceeds via a novel mechanism in which the DNA glycosylase NEIL3 unhooks the ICL. Here we examined the ability of the glycosylase domain of murine NEIL3 (MmuNEIL3-GD) to unhook dA-AP ICLs. The enzyme selectively unhooks the dA-AP ICL located at the duplex/single-strand junction of splayed duplexes that model the strand-separated DNA at the leading edge of a replication fork. We show that the ability to unhook the dA-AP ICL is a specialized function of NEIL3 as this activity is not observed in other BER enzymes. Importantly, NEIL3 only unhooks the dA-AP ICL when the AP residue is located on what would be the leading template strand of a model replication fork. The same specificity for the leading template strand was observed with a 5,6-dihydrothymine monoadduct, demonstrating that this preference is a general feature of the glycosylase and independent of the type of DNA damage. Overall, the results show that the glycosylase domain of NEIL3, lacking the C-terminal NPL4 and GRF zinc finger motifs, is competent to unhook the dA-AP ICL in splayed substrates and independently enforces important substrate preferences on the repair process. [ABSTRACT FROM AUTHOR]

• Pre-mRNA for the DNA repair enzyme NEIL1 is edited by adenosine deaminase ADAR1. • The efficiencies of unedited (K242) and edited (K242R) NEIL1 were compared. • K242 was more efficient in excision of ThyGly and aflatoxin-induced FapyGua lesions. • K242R was more efficient in excision of FapyAde, FapyGua, and 5-OH-Cyt lesions. Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid change of lysine 242 to arginine. Previous investigations of the catalytic efficiencies of the two forms of the enzyme revealed differential release of thymine glycol (ThyGly) from synthetic oligodeoxynucleotides, with the unedited form, NEIL1 K242 being ≈30-fold more efficient than the edited NEIL1 K242R. In contrast, when these enzymes were reacted with oligodeoxynucleotides containing guanidinohydantoin or spiroiminohydantoin, the edited K242R form was ≈3-fold more efficient than the unedited NEIL1. However, no prior studies have investigated the efficiencies of these two forms of NEIL1 on either high-molecular weight DNA containing multiple oxidatively-induced base damages, or oligodeoxynucleotides containing a bulky alkylated formamidopyrimidine. To understand the extent of changes in substrate recognition, γ-irradiated calf thymus DNA was treated with either edited or unedited NEIL1 and the released DNA base lesions analyzed by gas chromatography-tandem mass spectrometry. Of all the measured DNA lesions, imidazole ring-opened 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were preferentially released by both NEIL1 enzymes with K242R being ≈1.3 and 1.2-fold more efficient than K242 on excision of FapyAde and FapyGua, respectively. Consistent with the prior literature, large differences (≈7.5 to 12-fold) were measured in the excision of ThyGly from genomic DNA by the unedited versus edited NEIL1. In contrast, the edited NEIL1 was more efficient (≈3 to 5-fold) on release of 5-hydroxycytosine. Excision kinetics on DNA containing a site-specific aflatoxin B 1 -FapyGua adduct revealed an ≈1.4-fold higher rate by the unedited NEIL1. Molecular modeling provides insight into these differential substrate specificities. The results of this study and in particular, the comparison of substrate specificities of unedited and edited NEIL1 using biologically and clinically important base lesions, are critical for defining its role in preservation of genomic integrity. [ABSTRACT FROM AUTHOR]