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XPA (Xeroderma pigmentosum complementation group A) is a core scaffold protein that plays significant roles in DNA damage verification and recruiting downstream endonucleases in the nucleotide excision repair (NER) pathway. Here, we present the 2.81 Å resolution crystal structure of the DNA-binding domain (DBD) of human XPA in complex with an undamaged splayed-arm DNA substrate with a single pair of non-complementary nucleotides. The structure reveals that two XPA molecules bind to one splayed-arm DNA with a 10-bp duplex recognition motif in a non-sequence-specific manner. XPA molecules bind to both ends of the DNA duplex region with a characteristic β-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic β-hairpin. Upon DNA binding, the C-terminal last helix of XPA would shift towards the minor groove of the DNA substrate for better interaction. Notably, human XPA is able to bind to the undamaged DNA duplex without any kinks, and XPA-DNA binding does not bend the DNA substrate obviously. This study provides structural basis for the binding mechanism of XPA to the undamaged splayed-arm DNA with a single pair of non-complementary nucleotides., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The number of DNA polymerases identified in each organism has mushroomed in the past two decades. Most newly found DNA polymerases specialize in translesion synthesis and DNA repair instead of replication. Although intrinsic error rates are higher for translesion and repair polymerases than for replicative polymerases, the specialized polymerases increase genome stability and reduce tumorigenesis. Reflecting the numerous types of DNA lesions and variations of broken DNA ends, translesion and repair polymerases differ in structure, mechanism, and function. Here, we review the unique and general features of polymerases specialized in lesion bypass, as well as in gap-filling and end-joining synthesis.

Genomic instability in disease and its fidelity in health depend on the DNA damage response (DDR), regulated in part from the complex of meiotic recombination 11 homolog 1 (MRE11), ATP-binding cassette-ATPase (RAD50), and phosphopeptide-binding Nijmegen breakage syndrome protein 1 (NBS1). The MRE11-RAD50-NBS1 (MRN) complex forms a multifunctional DDR machine. Within its network assemblies, MRN is the core conductor for the initial and sustained responses to DNA double-strand breaks, stalled replication forks, dysfunctional telomeres, and viral DNA infection. MRN can interfere with cancer therapy and is an attractive target for precision medicine. Its conformations change the paradigm whereby kinases initiate damage sensing. Delineated results reveal kinase activation, posttranslational targeting, functional scaffolding, conformations storing binding energy and enabling access, interactions with hub proteins such as replication protein A (RPA), and distinct networks at DNA breaks and forks. MRN biochemistry provides prototypic insights into how it initiates, implements, and regulates multifunctional responses to genomic stress.

B-Myb, a highly conserved member of the Myb transcription factor family, is expressed ubiquitously in proliferating cells and controls the cell cycle dependent transcription of G2/M-phase genes. Deregulation of B-Myb has been implicated in oncogenesis and loss of genomic stability. We have identified B-Myb as a novel interaction partner of the Mre11-Rad50-Nbs1 (MRN) complex, a key player in the repair of DNA double strand breaks. We show that B-Myb directly interacts with the Nbs1 subunit of the MRN complex and is recruited transiently to DNA-damage sites. In response to DNA-damage B-Myb is phosphorylated by protein kinase GSK3β and released from the MRN complex. A B-Myb mutant that cannot be phosphorylated by GSK3β disturbs the regulation of pro-mitotic B-Myb target genes and leads to inappropriate mitotic entry in response to DNA-damage. Overall, our work suggests a novel function of B-Myb in the cellular DNA-damage signalling., Competing Interests: The authors declare no competing financial interests.

Genome integrity is vital to cellular homeostasis and its forfeiture is linked to deleterious consequences-cancer, immunodeficiency, genetic disorders and premature aging. The human ubiquitin ligase Pso4/Prp19 has emerged as a critical component of multiple DNA damage response (DDR) signaling networks. It not only senses DNA damage, binds double-stranded DNA in a sequence-independent manner, facilitates processing of damaged DNA, promotes DNA end joining, regulates replication protein A (RPA2) phosphorylation and ubiquitination at damaged DNA, but also regulates RNA splicing and mitotic spindle formation in its integral capacity as a scaffold for a multimeric core complex. Accordingly, by virtue of its regulatory and structural interactions with key proteins critical for genome integrity-DNA double-strand break (DSB) repair, DNA interstrand crosslink repair, repair of stalled replication forks and DNA end joining-it fills a unique niche in restoring genomic integrity after multiple types of DNA damage and thus has a vital role in maintaining chromatin integrity and cellular functions. These properties may underlie its ability to thwart replicative senescence and, not surprisingly, have been linked to the self-renewal and colony-forming ability of murine hematopoietic stem cells. This review highlights recent advances in hPso4 research that provides a fascinating glimpse into the pleiotropic activities of a ubiquitously expressed multifunctional E3 ubiquitin ligase.

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a β-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

DNA damage in the form of abasic sites, chemically altered nucleotides, and strand fragmentation is the foremost limitation in obtaining genetic information from many ancient samples. Upon cell death, DNA continues to endure various chemical attacks such as hydrolysis and oxidation, but repair pathways found in vivo no longer operate. By incubating degraded DNA with specific enzyme combinations adopted from these pathways, it is possible to reverse some of the post-mortem nucleic acid damage prior to downstream analyses such as library preparation, targeted enrichment, and high-throughput sequencing. Here, we evaluate the performance of two available repair protocols on previously characterized DNA extracts from four mammoths. Both methods use endonucleases and glycosylases along with a DNA polymerase-ligase combination. PreCR Repair Mix increases the number of molecules converted to sequencing libraries, leading to an increase in endogenous content and a decrease in cytosine-to-thymine transitions due to cytosine deamination. However, the effects of Nelson Repair Mix on repair of DNA damage remain inconclusive.

The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. In this study, two types of DNA binding assays were used for the detailed analysis of interaction of these proteins with damaged DNA. An electrophoretic mobility shift assay revealed that human and yeast orthologs behave similarly in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed using fluorescent depolarization measurements. The XPC-RAD23B and the Rad4-Rad23 proteins bind to the damaged 15 nt bubble-DNA structure mimicking in size the "transcription bubble" DNA intermediate with the highest affinity (KD values ~10(-10)  M or less) that is reduced in the following order: damaged bubble > undamaged bubble > damaged duplex > undamaged duplex. The affinity of XPC/Rad4 for various DNAs was shown to correlate with DNA bending angle. The results obtained show clearly that more deviation from regular DNA structure leads to higher XPC/Rad4 affinity., (Copyright © 2013 John Wiley & Sons, Ltd.)

Poly-ADP-ribosylation is a unique post-translational modification participating in many biological processes, such as DNA damage response. Here, we demonstrate that a set of Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains recognizes poly(ADP-ribose) (PAR) both in vitro and in vivo. Among these FHA and BRCT domains, the FHA domains of APTX and PNKP interact with iso-ADP-ribose, the linkage of PAR, whereas the BRCT domains of Ligase4, XRCC1, and NBS1 recognize ADP-ribose, the basic unit of PAR. The interactions between PAR and the FHA or BRCT domains mediate the relocation of these domain-containing proteins to DNA damage sites and facilitate the DNA damage response. Moreover, the interaction between PAR and the NBS1 BRCT domain is important for the early activation of ATM during DNA damage response and ATM-dependent cell cycle checkpoint activation. Taken together, our results demonstrate two novel PAR-binding modules that play important roles in DNA damage response.

Exogenous chemicals may produce DNA methylation that is potentially toxic to living systems. Methylated DNA bases are difficult to detect with biosensors because the methyl group is small and chemically inert. In this report, a label-free photoelectrochemical sensor was developed for the selective detection of chemically methylated bases in DNA films. The sensor employed two DNA repair enzymes, human alkyladenine DNA glycosylase and human apurinic/apyrimidinic endonuclease, to convert DNA methylation sites in DNA films on indium tin oxide electrodes into strand breaks. A DNA intercalator, Ru(bpy)2(dppz)(2+) (bpy=2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) was then used as the photoelectrochemical signal indicator to detect the DNA strand breaks. Its photocurrent signal was found to correlate inversely with the amount of 3-methyladenines (metAde) produced with a methylating agent, methylmethane sulfonate (MMS). The sensor detected the methylated bases produced with as low as 1 mM MMS, at which concentration the amount of metAde on the sensor surface was estimated to be 0.5 pg, or 1 metAde in 1.6 × 10(5) normal bases. Other DNA base modification products, such as 5-methylcytosine and DNA adducts with ethyl and styrene groups did not attenuate the photocurrent, demonstrating good selectivity of the sensor. This strategy can be utilized to develop sensors for the detection of other modified DNA bases with specific DNA repair enzymes.

The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. The interaction of these proteins with damaged DNA was analyzed using model DNA duplexes containing a single fluorescein-substituted dUMP analog as a lesion. An electrophoretic mobility shift assay revealed similarity between human and yeast proteins in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed by fluorescent depolarization measurements. XPC-RAD23B and Rad4-Rad23 proteins demonstrate approximately equal binding affinity to the damaged DNA duplex (K(D) ∼ (0.5 ± 0.1) and (0.6 ± 0.3) nM, respectively). Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged DNA was shown. Under conditions of equimolar binding to DNA both proteins exhibited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide. The positioning of the XPC and Rad4 proteins on damaged DNA by photocross-linking footprinting is consistent with x-ray analysis of the Rad4-DNA crystal complex. The identity of the XPC and Rad4 location illustrates the common principles of structure organization of DNA damage-scanning proteins from different Eukarya organisms.

Repairing damaged DNA is essential for an organism's survival. UV damage endonuclease (UVDE) is a DNA-repair enzyme that can recognize and incise different types of damaged DNA. We present the structure of Sulfolobus acidocaldarius UVDE on its own and in a pre-catalytic complex with UV-damaged DNA containing a 6-4 photoproduct showing a novel 'dual dinucleotide flip' mechanism for recognition of damaged dipyrimidines: the two purines opposite to the damaged pyrimidine bases are flipped into a dipurine-specific pocket, while the damaged bases are also flipped into another cleft.

ALKBH2 is a direct DNA repair dioxygenase guarding the mammalian genome against N(1)-methyladenine, N(3)-methylcytosine and 1,N(6)-ethenoadenine damage. A prerequisite for repair is to identify these lesions in the genome. Here we present crystal structures of human ALKBH2 bound to different duplex DNAs. Together with computational and biochemical analyses, our results suggest that DNA interrogation by ALKBH2 has two previously unknown features: (i) ALKBH2 probes base-pair stability and detects base pairs with reduced stability, and (ii) ALKBH2 does not have nor need a damage-checking site, which is critical for preventing spurious base cleavage for several glycosylases. The demethylation mechanism of ALKBH2 insures that only cognate lesions are oxidized and reversed to normal bases, and that a flipped, non-substrate base remains intact in the active site. Overall, the combination of duplex interrogation and oxidation chemistry allows ALKBH2 to detect and process diverse lesions efficiently and correctly.

UV-sensitive syndrome (UV(S)S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV(S)S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV(S)S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS , one of whom is described for the first time here, formed a separate UV(S)S-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV(S)S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders.

Three teams have applied whole-exome and proteome methods to identify a new cofactor of human RNA polymerase II that is required for the recovery of transcription on damaged templates. The identification of this new factor raises questions about the causal relationships between molecular mechanisms of transcription regulation and excision repair and developmental and neurological disease and nonmalignant skin photosensitivity.

Nucleotide excision repair (NER) is the most versatile DNA-repair pathway in all organisms. While bacteria require only three proteins to complete the incision step of NER, eukaryotes employ about 30 proteins to complete the same step. Here we summarize recent studies demonstrating that ubiquitination, a post-translational modification, plays critical roles in regulating the NER activity either dependent on or independent of ubiquitin-proteolysis. Several NER components have been shown as targets of ubiquitination while others are actively involved in the ubiquitination process. We argue through this analysis that ubiquitination serves to coordinate various steps of NER and meanwhile connect NER with other related pathways to achieve the efficient global DNA-damage response.

Editing of the pre-mRNA for the DNA repair enzyme NEIL1 causes a lysine to arginine change in the lesion recognition loop of the protein. The two forms of NEIL1 are shown here to have distinct enzymatic properties. The edited form removes thymine glycol from duplex DNA 30 times more slowly than the form encoded in the genome, whereas editing enhances repair of the guanidinohydantoin lesion by NEIL1. In addition, we show that the NEIL1 recoding site is a preferred editing site for the RNA editing adenosine deaminase ADAR1. The edited adenosine resides in an A-C mismatch in a hairpin stem formed by pairing of exon 6 to the immediate upstream intron 5 sequence. As expected for an ADAR1 site, editing at this position is increased in human cells treated with interferon α. These results suggest a unique regulatory mechanism for DNA repair and extend our understanding of the impact of RNA editing.

N(1)-meA and N(3)-meC are cytotoxic DNA base methylation lesions that can accumulate in the genomes of various organisms in the presence of S(N)2 type methylating agents. We report here the structural characterization of these base lesions in duplex DNA using a cross-linked protein-DNA crystallization system. The crystal structure of N(1)-meA:T pair shows an unambiguous Hoogsteen base pair with a syn conformation adopted by N(1)-meA, which exhibits significant changes in the opening, roll and twist angles as compared to the normal A:T base pair. Unlike N(1)-meA, N(3)-meC does not establish any interaction with the opposite G, but remains partially intrahelical. Also, structurally characterized is the N(6)-meA base modification that forms a normal base pair with the opposite T in duplex DNA. Structural characterization of these base methylation modifications provides molecular level information on how they affect the overall structure of duplex DNA. In addition, the base pairs containing N(1)-meA or N(3)-meC do not share any specific characteristic properties except that both lesions create thermodynamically unstable regions in a duplex DNA, a property that may be explored by the repair proteins to locate these lesions.

Human DNA polymerase N (PolN) is an A-family nuclear DNA polymerase whose function is unknown. This study examines the possible role of PolN in DNA repair in human cells treated with PolN-targeted siRNA. HeLa cells with siRNA-mediated knockdown of PolN were more sensitive than control cells to DNA cross-linking agent mitomycin C (MMC) but were not hypersensitive to UV irradiation. The MMC hypersensitivity of PolN knockdown cells was rescued by the overexpression of DNA polymerase-proficient PolN but not by DNA polymerase-deficient PolN. Furthermore, in vitro experiments showed that purified PolN conducts low-efficiency nonmutagenic bypass of a psoralen DNA interstrand cross-link (ICL), whose structure resembles an intermediate in the proposed pathway of ICL repair. These results suggest that PolN might play a role in translesion DNA synthesis during ICL repair in human cells.

Base excision repair (BER) is the primary DNA repair pathway that corrects base lesions that arise due to oxidative, alkylation, deamination, and depurinatiation/depyrimidination damage. BER facilitates the repair of damaged DNA via two general pathways - short-patch and long-patch. The shortpatch BER pathway leads to a repair tract of a single nucleotide. Alternatively, the long-patch BER pathway produces a repair tract of at least two nucleotides. The BER pathway is initiated by one of many DNA glycosylases, which recognize and catalyze the removal of damaged bases. The completion of the BER pathway is accomplished by the coordinated action of at least three additional enzymes. These downstream enzymes carry out strand incision, gap-filling and ligation. The high degree of BER conservation between E. coli and mammals has lead to advances in our understanding of mammalian BER. This review will provide a general overview of the mammalian BER pathway. (Part of a Multi-author Review).

THE FOREIGN LESION: The mechanistic questions for DNA base damage detection by repair proteins are discussed in this Minireview. Repair proteins could either probe and locate a weakened base pair that results from base damage, or passively capture an extrahelical base lesion in the first step of damage searching on double-stranded DNA. How some repair proteins, such as AGT (see figure), locate base lesions in DNA is still not fully understood.To remove a few damaged bases efficiently from the context of the entire genome, the DNA base repair proteins rely on remarkably specific detection mechanisms to locate base lesions. This efficient molecular recognition event inside cells has been extensively studied with various structural and biochemical tools. These studies suggest that DNA base damage can be located by repair proteins by using two mechanisms: a repair protein can probe and detect a weakened base pair that results from mutagenic or cytotoxic base damage; alternatively, a protein can passively capture and stabilize an extrahelical base lesion. Our chemical and structural studies on the direct DNA repair proteins hAGT, C-Ada and ABH2 suggest that these proteins search for weakened base pairs in their first step of damage searching. We have also discovered a very unique base-flipping mechanism used by the DNA repair protein AlkB. This protein distorts DNA and favors single stranded DNA (ssDNA) substrates over double-stranded (dsDNA) ones. Potentially, it locates base lesions in dsDNA by imposing a constraint that targets less rigid regions of the duplex DNA. The exact mechanism of how AlkB and related proteins search for damage in ssDNA and dsDNA still awaits further studies.

Accumulation of genetic changes due to the presence of unrepaired DNA lesions can lead to cancer development and other diseases. Nucleic acid modifications also play key roles in many essential life processes. We have developed a series of chemically modified nucleic acid analogues that can be applied to stabilize protein-nucleic acid interactions for structural and proteomic studies. Some of the probes have also been employed to study nucleic acid-nucleic acid interactions.

Nucleotide excision repair is a cut-and-patch pathway that eliminates potentially mutagenic DNA lesions caused by ultraviolet light, electrophilic chemicals, oxygen radicals and many other genetic insults. Unlike antigen recognition by the immune system, which employs billions of immunoglobulins and T-cell receptors, the nucleotide excision repair complex relies on just a few generic factors to detect an extremely wide range of DNA adducts. This molecular versatility is achieved by a bipartite strategy initiated by the detection of abnormal strand fluctuations, followed by the localization of injured residues through an enzymatic scanning process coupled to DNA unwinding. The early recognition subunits are able to probe the thermodynamic properties of nucleic acid substrates but avoid direct contacts with chemically altered bases. Only downstream subunits of the bipartite recognition process interact more closely with damaged bases to delineate the sites of DNA incision. Thus, consecutive factors expand the spectrum of deleterious genetic lesions conveyed to DNA repair by detecting distinct molecular features of target substrates.

Single-base lesions in DNA are repaired predominantly by base excision repair (BER). DNA polymerase beta (pol beta) is the polymerase of choice in the preferred single-nucleotide BER pathway. The characteristic phenotype of mouse fibroblasts with a deletion of the pol beta gene is moderate hypersensitivity to monofunctional alkylating agents, e.g., methyl methanesulfonate (MMS). Increased sensitivity to MMS is also seen in the absence of pol beta partner proteins XRCC1 and PARP-1, and under conditions where BER efficiency is reduced by synthetic inhibitors. PARP activity plays a major role in protection against MMS-induced cytotoxicity, and cells treated with a combination of non-toxic concentrations of MMS and a PARP inhibitor undergo cell cycle arrest and die by a Chk1-dependent apoptotic pathway. Since BER-deficient cells and tumors are similarly hypersensitive to the clinically used chemotherapeutic methylating agent temozolomide, modulation of DNA damage-induced cell signaling pathways, as well as BER, are attractive targets for potentiating chemotherapy.

Three-dimensional structures of DNA N-glycosylases and N-glycosylase/apyrimidine/apurine (AP)-lyase enzymes and other critical components of base excision repair (BER) machinery including structure-specific nuclease, repair polymerase, DNA ligase, and PCNA tethering complexes reveal the overall unity of the simple cut and patch process of DNA repair for damaged bases. In general, the damage-specific excision is initiated by structurally-variable DNA glycosylases targeted to distinct base lesions. This committed excision step is followed by a subsequent damage-general processing of the resulting abasic sites and 3' termini, the insertion of the correct base by a repair DNA polymerase, and finally sealing the nicked backbone by DNA ligase. However, recent structures of protein-DNA and protein-protein complexes and other BER machinery are providing a more in-depth look into the intricate functional diversity and complexity of maintaining genomic integrity despite very high levels of constant DNA base damage from endogenous as well as environmental agents. Here we focus on key discoveries concerning BER structural biology that speak to better understanding the damage recognition, reaction mechanisms, conformational controls, coordinated handoffs, and biological activities including links to cancer. As the three-dimensional crystal and NMR structures for the protein and DNA complexes of all major components of the BER system have now been determined, we provide here a relatively complete description of the key complexes starting from DNA base damage detection and excision to the final ligation process. As our understanding of BER structural biology and the molecular basis for cancer improve, we predict that there will be multiple links joining BER enzyme mutations and cancer predispositions, such as now seen for MYH. Currently, structural results are realizing the promise that high-resolution structures provide detailed insights into how these BER proteins function to specifically recognize, remove, and repair DNA base damage without the release of toxic and mutagenic intermediates.

Oxidatively induced DNA lesions have been implicated in the etiology of many diseases (including cancer) and in aging. Repair of oxidatively damaged bases in all organisms occurs primarily via the DNA base excision repair (BER) pathway, initiated with their excision by DNA glycosylases. Only two mammalian DNA glycosylases, OGG1 and NTH1 of E. coli Nth family, were previously characterized, which excise majority of the oxidatively damaged base lesions. We recently discovered and characterized two human orthologs of E. coli Nei, the prototype of the second family of oxidized base-specific glycosylases and named them NEIL (Nei-like)-1 and 2. NEILs are distinct from NTH1 and OGG1 in structural features and reaction mechanism but act on many of the same substrates. Nth-type DNA glycosylases after base excision, cleave the DNA strand at the resulting AP-site to produce a 3'-alphabeta unsaturated aldehyde whereas Nei-type enzymes produce 3'-phosphate terminus. E. coli APEs efficiently remove both types of termini in addition to cleaving AP sites to generate 3'-OH, the primer terminus for subsequent DNA repair synthesis. In contrast, the mammalian APE, APE1, which has an essential role in NTH1/OGG1-initiated BER, has negligible 3'-phosphatase activity and is dispensable for NEIL-initiated BER. Polynucleotide kinase (PNK), present in mammalian cells but not in E. coli, removes the 3' phosphate, and is involved in NEIL-initiated BER. NEILs show a unique preference for excising lesions from a DNA bubble, while most DNA glycosylases, including OGG1 and NTH1, are active only with duplex DNA. The dichotomy in the preference of NEILs and NTH1/OGG1 for bubble versus duplex DNA substrates suggests that NEILs function preferentially in repair of base lesions during replication and/or transcription and hence play a unique role in maintaining the functional integrity of mammalian genomes.

The DNA damage response, triggered by DNA replication stress or DNA damage, involves the activation of DNA repair and cell cycle regulatory proteins including the MRN (Mre11, Rad50, and Nbs1) complex and replication protein A (RPA). The induction of replication stress by hydroxyurea (HU) or DNA damage by camptothecin (CAMPT), etoposide (ETOP), or mitomycin C (MMC) led to the formation of nuclear foci containing phosphorylated Nbs1. HU and CAMPT treatment also led to the formation of RPA foci that co-localized with phospho-Nbs1 foci. After ETOP treatment, phospho-Nbs1 and RPA foci were detected but not within the same cell. MMC treatment resulted in phospho-Nbs1 foci formation in the absence of RPA foci. Consistent with the presence or absence of RPA foci, RPA hyperphosphorylation was present following HU, CAMPT, and ETOP treatment but absent following MMC treatment. The lack of co-localization of phospho-Nbs1 and RPA foci may be due to relatively shorter stretches of single-stranded DNA generated following ETOP and MMC treatment. These data suggest that, even though the MRN complex and RPA can interact, their interaction may be limited to responses to specific types of lesions, particularly those that have longer stretches of single-stranded DNA. In addition, the consistent formation of phospho-Nbs1 foci in all of the treatment groups suggests that the MRN complex may play a more universal role in the recognition and response to DNA lesions of all types, whereas the role of RPA may be limited to certain subsets of lesions.

Free radical attack on the sugar-phosphate backbone generates oxidized apurinic/apyrimidinic (AP) residues in DNA. 2'-deoxyribonolactone (dL) is a C1'-oxidized AP site damage generated by UV and gamma-irradiation, and certain anticancer drugs. If not repaired dL produces G-->A transitions in Escherichia coli. In the base excision repair (BER) pathway, AP endonucleases are the major enzymes responsible for 5'-incision of the regular AP site (dR) and dL. DNA glycosylases with associated AP lyase activity can also efficiently cleave regular AP sites. Here, we report that dL is a substrate for AP endonucleases but not for DNA glycosylases/AP lyases. The kinetic parameters of the dL-incision were similar to those of the dR. DNA glycosylases such as E. coli formamidopyrimidine-DNA glycosylase, mismatch-specific uracil-DNA glycosylase, and human alkylpurine-DNA N-glycosylase bind strongly to dL without cleaving it. We show that dL cross-links with the human proteins 8-oxoguanine-DNA (hOGG1) and thymine glycol-DNA glycosylases (hNth1), and dR cross-links with Nth and hNth1. These results suggest that dL and dR induced genotoxicity might be strengthened by BER pathway in vivo.

Mammalian rpS3, a ribosomal protein S3 with a DNA repair endonuclease activity, nicks heavily UV-irradiated DNA and DNA containing AP sites. RpS3 calls for a novel endonucleolytic activity on AP sites generated from pyrimidine dimers by T4 pyrimidine dimer glycosylase activity. This study revealed that rpS3 cleaves the lesions including AP sites, thymine glycols, and other UV damaged lesions such as pyrimidine dimers. This enzyme does not have a glycosylase activity as predicted from its amino acid sequence. However, it has an endonuclease activity on DNA containing thymine glycol, which is exactly overlapped with UV-irradiated or AP DNAs, indicating that rpS3 cleaves phosphodiester bonds of DNAs containing altered bases with broad specificity acting as a base-damage-endonuclease. RpS3 cleaves supercoiled UV damaged DNA more efficiently than the relaxed counterpart, and the endonuclease activity of rpS3 was inhibited by MgCl2 on AP DNA but not on UV-irradiated DNA.

The discovery of the recA gene toward the middle of the 20th century sparked work in perhaps one of the most biochemically and biophysically intriguing systems of DNA repair-homologous recombination. The inner workings of this system, in particular those of the more complex eukaryotes, have been and in many ways remain mysterious. Yet at the turn of this century, a wealth of structural and genetic results has unveiled a detailed picture of the roles, relationships, and mechanics of interacting homologous recombination proteins. Here we focus on the predominant questions addressed by these exciting 21st century structural results-from detection of broken DNA ends to coordination of pathway progression. The emerging structural view of double-strand break repair, therefore, reveals the molecular basis both for functions specific to DNA recombination and for general features characterizing DNA repair processes.