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Ionizing radiation (IR) causes DNA damage, particularly DNA double-strand breaks (DSBs), which have significant implications for genome stability. The major pathways of repairing DSBs are homologous recombination (HR) and nonhomologous end joining (NHEJ). However, the repair mechanism of IR-induced DSBs in embryos is not well understood, despite extensive research in somatic cells. The externally developing aquatic organism, Xenopus tropicalis, serves as a valuable model for studying embryo development. A significant increase in zygotic transcription occurs at the midblastula transition (MBT), resulting in a longer cell cycle and asynchronous cell divisions. This study examines the impact of X-ray irradiation on Xenopus embryos before and after the MBT. The findings reveal a heightened X-ray sensitivity in embryos prior to the MBT, indicating a distinct shift in the DNA repair pathway during embryo development. Importantly, we show a transition in the dominant DSB repair pathway from NHEJ to HR before and after the MBT. These results suggest that the MBT plays a crucial role in altering DSB repair mechanisms, thereby influencing the IR sensitivity of developing embryos., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

DNA-protein cross-links (DPCs) are generated by internal factors such as cellular aldehydes that are generated during normal metabolism and external factors such as environmental mutagens. A nucleoside analog, 5-aza-2'-deoxycytidine (5-azadC), is randomly incorporated into the genome during DNA replication and binds DNA methyltransferase 1 (DNMT1) covalently to form DNMT1-DPCs without inducing DNA strand breaks. Despite the recent progress in understanding the mechanisms of DPCs repair, how DNMT1-DPCs are repaired is unclear. The metalloprotease SPRTN has been considered as the primary enzyme to degrade protein components of DPCs to initiate the repair of DPCs. In this study, we showed that SPRTN-deficient ( SPRTN -/- ) human TK6 cells displayed high sensitivity to 5-azadC, and the removal of 5-azadC-induced DNMT1-DPCs was significantly slower in SPRTN -/- cells than that in wild-type cells. We also showed that the ubiquitination-dependent proteasomal degradation, which was independent of the SPRTN-mediated processing, was also involved in the repair of DNMT1-DPCs. Unexpectedly, we found that cells that are double deficient in tyrosyl DNA phosphodiesterase 1 and 2 ( TDP1 -/- TDP2 -/- ) were also sensitive to 5-azadC, although the removal of 5-azadC-induced DNMT1-DPCs was not compromised significantly. Furthermore, the 5-azadC treatment induced a marked accumulation of chromosomal breaks in SPRTN -/- as well as TDP1 -/- TDP2 -/- cells compared to wild-type cells, strongly suggesting that the 5-azadC-induced cell death was attributed to chromosomal DNMT1-DPCs. We conclude that SPRTN protects cells from 5-azadC-induced DNMT1-DPCs, and SPRTN may play a direct proteolytic role against DNMT1-DPCs and TDP1/TDP2 also contributes to suppress genome instability caused by 5-azadC in TK6 cells.

Topoisomerase I (TOP1) resolves DNA topology during replication and transcription. The enzyme forms an intermediate TOP1 cleavage complex (TOP1cc) through transient TOP1-DNA-protein crosslinks. Camptothecin is a frontline anticancer agent that freezes this reaction intermediate, leading to the generation of irreversible TOP1ccs that act as 3'-blocking lesions. It is widely accepted that TOP1cc is repaired via a two-step pathway involving proteasomal degradation of TOP1cc to the crosslinked peptide, followed by removal of the TOP1cc-derived peptide from DNA by tyrosyl-DNA phosphodiesterase 1 (TDP1). In the present study, we developed an assay system to estimate repair kinetics of TOP1cc separately in the first and second steps, using monoclonal antibodies against the TOP1 protein and the TOP1 catalytic site peptide-DNA complex, respectively. Although TDP1-deficient (TDP1 -/- ) TK6 cells had normal kinetics of the first step, a delay in the kinetics of the second step was observed relative to that in wild-type cells. Tyrosyl-DNA phosphodiesterase 2 (TDP2) reportedly promotes the repair of TOP1-induced DNA damage in the absence of TDP1. The present assays additionally demonstrated that TDP2 promotes the second, but not the first, step of TOP1cc repair in the absence of TDP1. We also analyzed sensitivities of TK6 cells with deficiencies in TDP1 and/or TDP2 to agents that produce 3' -blocking lesions. These experiments showed that TDP1 -/- TDP2 -/- cells were more sensitive to the agents Azidothymidine (zidovudine), Cytarabine, Abacavir, Gemcitabine, and Trifluridine than TDP1 -/- or TDP2 -/- cells. Taken together, our findings confirm the roles of TDP2 in the repair of 3'-blocking lesions., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Proteins are covalently trapped on DNA to form DNA-protein cross-links (DPCs) when cells are exposed to DNA-damaging agents. Aldehyde compounds produce common types of DPCs that contain proteins in an undisrupted DNA strand. Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs topoisomerase 1 (TOPO1) that is trapped at the 3'-end of DNA. In the present study, we examined the contribution of TDP1 to the repair of formaldehyde-induced DPCs using a reverse genetic strategy with chicken DT40 cells. The results obtained showed that cells deficient in TDP1 were sensitive to formaldehyde. The removal of formaldehyde-induced DPCs was slower in tdp1-deficient cells than in wild type cells. We also found that formaldehyde did not produce trapped TOPO1, indicating that trapped TOPO1 was not a primary cytotoxic DNA lesion that was generated by formaldehyde and repaired by TDP1. The formaldehyde treatment resulted in the accumulation of chromosomal breakages that were more prominent in tdp1-deficient cells than in wild type cells. Therefore, TDP1 plays a critical role in the repair of formaldehyde-induced DPCs that are distinct from trapped TOPO1., Competing Interests: The authors have declared that no competing interests exist.

DNA is associated with proteins that are involved in its folding and transaction processes. When cells are exposed to chemical cross-linking agents or free radical-generating ionizing radiation, DNA-associated proteins are covalently trapped within the DNA to produce DNA-protein cross-links (DPCs). DPCs produced by these agents contain cross-linked proteins in an undisrupted DNA strand. Some DNA-metabolizing enzymes that form covalent reaction intermediates can also be irreversibly trapped in the presence of inhibitors or DNA damage to give rise to abortive DPCs. The abortive DPCs often contain cross-linked proteins attached to the 5' or 3' end of a DNA strand break. In vitro studies show that steric hindrance caused by cross-linked proteins impedes the progression of DNA helicases and polymerases and of RNA polymerases. The modes and consequences by which DPCs impede replication and transcription processes are considerably different from those with conventional DNA lesions. Thus, DPCs are formidable challenges to maintaining genome integrity and faithful gene expression. Current models of DPC repair involve direct and indirect removal of DPCs. The direct mechanism works for DPCs that contain topoisomerase 2 attached to the 5' end of DNA. The Mre11-Rad50-Nbs1 complex cleaves the site internal to the DPC and directly releases a DPC-containing oligonucleotide. The indirect mechanism involves degradation of cross-linked proteins by proteasomes or the recently identified DPC proteases Wss1 and Sprtn to relieve steric hindrance of DPCs. The resulting peptide-cross-links might be processed by translesion synthesis or other canonical repair mechanisms: however, the exact mechanism remains to be elucidated., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ionizing radiation produces various DNA lesions such as base damage, DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-protein cross-links (DPCs). Of these, the biological significance of DPCs remains elusive. In this article, we focus on radiation-induced DPCs and review the current understanding of their induction, properties, repair, and biological consequences. When cells are irradiated, the formation of base damage, SSBs, and DSBs are promoted in the presence of oxygen. Conversely, that of DPCs is promoted in the absence of oxygen, suggesting their importance in hypoxic cells, such as those present in tumors. DNA and protein radicals generated by hydroxyl radicals (i.e., indirect effect) are responsible for DPC formation. In addition, DPCs can also be formed from guanine radical cations generated by the direct effect. Actin, histones, and other proteins have been identified as cross-linked proteins. Also, covalent linkages between DNA and protein constituents such as thymine-lysine and guanine-lysine have been identified and their structures are proposed. In irradiated cells and tissues, DPCs are repaired in a biphasic manner, consisting of fast and slow components. The half-time for the fast component is 20min-2h and that for the slow component is 2-70h. Notably, radiation-induced DPCs are repaired more slowly than DSBs. Homologous recombination plays a pivotal role in the repair of radiation-induced DPCs as well as DSBs. Recently, a novel mechanism of DPC repair mediated by a DPC protease was reported, wherein the resulting DNA-peptide cross-links were bypassed by translesion synthesis. The replication and transcription of DPC-bearing reporter plasmids are inhibited in cells, suggesting that DPCs are potentially lethal lesions. However, whether DPCs are mutagenic and induce gross chromosomal alterations remains to be determined., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aldehydes are genotoxic and cytotoxic molecules and have received considerable attention for their associations with the pathogenesis of various human diseases. In addition, exposure to anthropogenic aldehydes increases human health risks. The general mechanism of aldehyde toxicity involves adduct formation with biomolecules such as DNA and proteins. Although the genotoxic effects of aldehydes such as mutations and chromosomal aberrations are directly related to DNA damage, the role of DNA damage in the cytotoxic effects of aldehydes is poorly understood because concurrent protein damage by aldehydes has similar effects. In this study, we have analysed how saturated and α,β-unsaturated aldehydes exert cytotoxic effects through DNA and protein damage. Interestingly, DNA repair is essential for alleviating the cytotoxic effect of weakly toxic aldehydes such as saturated aldehydes but not highly toxic aldehydes such as long α,β-unsaturated aldehydes. Thus, highly toxic aldehydes inactivate cells exclusively by protein damage. Our data suggest that DNA interstrand crosslinks, but not DNA-protein crosslinks and DNA double-strand breaks, are the critical cytotoxic DNA damage induced by aldehydes. Further, we show that the depletion of intracellular glutathione and the oxidation of thioredoxin 1 partially account for the DNA damage-independent cytotoxicity of aldehydes. On the basis of these findings, we have proposed a mechanistic model of aldehyde cytotoxicity mediated by DNA and protein damage., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3'-OH and 5'-deoxyribose phosphate (5'-dRp) termini. DNA polymerase β (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5'-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER., (© The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Clustered DNA damage is a specific type of DNA damage induced by ionizing radiation. Any type of ionizing radiation traverses the target DNA molecule as a beam, inducing damage along its track. Our previous study showed that clustered DNA damage yields decreased with increased linear energy transfer (LET), leading us to investigate the importance of clustered DNA damage in the biological effects of heavy ion beam radiation. In this study, we analyzed the yield of clustered base damage (comprising multiple base lesions) in cultured cells irradiated with various heavy ion beams, and investigated isolated base damage and the repair process in post-irradiation cultured cells. Chinese hamster ovary (CHO) cells were irradiated by carbon, silicon, argon and iron ion beams with LETs of 13, 55, 90 and 200 keV µm(-1), respectively. Agarose gel electrophoresis of the cells with enzymatic treatments indicated that clustered base damage yields decreased as the LET increased. The aldehyde reactive probe procedure showed that isolated base damage yields in the irradiated cells followed the same pattern. To analyze the cellular base damage process, clustered DNA damage repair was investigated using DNA repair mutant cells. DNA double-strand breaks accumulated in CHO mutant cells lacking Xrcc1 after irradiation, and the cell viability decreased. On the other hand, mouse embryonic fibroblast (Mef) cells lacking both Nth1 and Ogg1 became more resistant than the wild type Mef. Thus, clustered base damage seems to be involved in the expression of heavy ion beam biological effects via the repair process., (© The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5'-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5'-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA-R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genomic DNA is associated with various structural, regulatory, and transaction proteins. The dynamic and reversible association between proteins and DNA ensures the accurate expression and propagation of genetic information. However, various endogenous, environmental, and chemotherapeutic agents induce DNA-protein crosslinks (DPCs), and hence covalently trap proteins on DNA. Since DPCs are extremely large compared to conventional DNA lesions, they probably impair many aspects of DNA transactions such as replication, transcription, and repair due to steric hindrance. Recent genetic and biochemical studies have shed light on the elaborate molecular mechanism by which cells repair or tolerate DPCs. This review summarizes the current knowledge regarding the repair and biochemical effects of the most ubiquitous form of DPCs, which are associated with no flanked DNA strand breaks. In bacteria small DPCs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed by RecBCD-dependent homologous recombination (HR). NER does not participate in the repair of DPCs in mammalian cells, since the upper size limit of DPCs amenable to mammalian NER is smaller than that of bacterial NER. Thus, DPCs are processed exclusively by HR. The reactivation of the stalled replication fork at DPCs by HR seems to involve fork breakage in mammalian cells but not in bacterial cells. In addition, recent proteomic studies have identified the numbers of proteins in DPCs induced by environmental and chemotherapeutic agents. However, it remains largely elusive how DPCs affect replication and transcription at the molecular level. Considering the extremely large nature of DPCs, it is possible that they impede the progression of replication and transcription machineries by mechanisms different from those for conventional DNA lesions. This might also be true for the DNA damage response and signaling mechanism., (2011 Elsevier B.V. All rights reserved.)

We have developed fluorescent probes for the detection of strand scission in the excision repair of oxidatively damaged bases. They were hairpin-shaped oligonucleotides, each containing an isomer of thymine glycol or 5,6-dihydrothymine as a damaged base in the center, with a fluorophore and a quencher at the 5'- and 3'-ends, respectively. Fluorescence was detected when the phosphodiester linkage at the damage site was cleaved by the enzyme, because the short fragment bearing the fluorophore could not remain in a duplex form hybridized to the rest of the molecule at the incubation temperature. The substrate specificities of Escherichia coli endonuclease III and its human homolog, NTH1, determined by using these probes agreed with those determined previously by gel electrophoresis using (32)P-labeled substrates. Kinetic parameters have also been determined by this method. Since different fluorophores were attached to the oligonucleotides containing each lesion, reactions with two types of substrates were analyzed separately in a single tube, by changing the excitation and detection wavelengths. These probes were degraded during an incubation with a cell extract. Therefore, phosphorothioate linkages were incorporated to protect the probes from nonspecific nucleases, and the base excision repair activity was successfully detected in HeLa cells.

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. We have recently shown that nucleotide excision repair (NER) and RecBCD-dependent homologous recombination (HR) collaboratively alleviate the lethal effect of DPCs in Escherichia coli. In this study, to gain further insight into the damage-processing mechanism for DPCs, we assessed the sensitivities of a panel of repair-deficient E. coli mutants to DPC-inducing agents, including formaldehyde (FA) and 5-azacytidine (azaC). We show here that the damage tolerance mechanism involving HR and subsequent replication restart (RR) provides the most effective means of cell survival against DPCs. Translesion synthesis does not serve as an alternative damage tolerance mechanism for DPCs in cell survival. Elimination of DPCs from the genome relies primarily on NER, which provides a second and moderately effective means of cell survival against DPCs. Interestingly, Cho rather than UvrC seems to be an effective nuclease for the NER of DPCs. Together with the genes responsible for HR, RR, and NER, the mutation of genes involved in several aspects of DNA repair and transactions, such as recQ, xth nfo, dksA, and topA, rendered cells slightly but significantly sensitive to FA but not azaC, possibly reflecting the complexity of DPCs or cryptic lesions induced by FA. UvrD may have an additional role outside NER, since the uvrD mutation conferred a slight azaC sensitivity on cells. Finally, DNA glycosylases mitigate azaC toxicity, independently of the repair of DPCs, presumably by removing 5-azacytosine or its degradation product from the chromosome.

Endogenous and environmental genotoxic agents produce DNA damage and induce cell death and mutations. The repair mechanisms of base lesions and single and double strand breaks have been well characterized in both prokaryotic and eukaryotic cells. However, the molecular pathways that repair or tolerate DNA-protein crosslinks (DPCs) remains to be largely elucidated. In this study, we constructed DNA substrates containing defined DPCs and assessed the incision activities of prokaryotic and eukaryotic nucleotide excision repair systems for DPCs in vitro.

DNA-protein crosslinks (DPCs) are extremely bulky DNA lesions, and steric hindrance imposed by covalently trapped proteins would hamper the transaction of DNA such as replication, transcription, and repair. However, it has been largely elusive how cells mitigate the genotoxic effect of DPCs. We have recently shown that nucleotide excision repair (NER) and homologous recombination (HR) differentially contribute to the repair of DPCs in E. coli cells. Several lines of genetic and biochemical evidence indicate that NER repairs DPCs with crosslinked proteins (CLPs) of sizes less than 12-14 kDa, whereas DPCs with oversized CLPs are processed exclusively by RecBCD-dependent HR. The present result shows that cells use the coordinated actions of NER and HR to deal with unusually bulky DNA lesions like DPCs.

TopBP1 is involved in DNA replication and DNA damage checkpoint. Recent studies have demonstrated that TopBP1 is a direct positive effecter of ATR. However, it is not known how TopBP1 recognizes damaged DNA. Here, we show that TopBP1 formed nuclear foci after exposure to ionizing radiation, but such TopBP1 foci were abolished in Nijmegen breakage syndrome cells. We also show that TopBP1 physically associated with NBS1 in vivo. These results suggested that NBS1 might regulate TopBP1 recruitment to the sites of DNA damage. TopBP1-depleted cells showed hypersensitivity to Mitomycin C and ionizing radiation, an increased frequency of sister-chromatid exchange level, and a reduced frequency of DNA double-strand break induced homologous recombination repair. Together, these results suggested that TopBP1 might be a mediator of DNA damage signaling from NBS1 to ATR and promote homologous recombination repair.

DNA-protein crosslinks (DPCs)-where proteins are covalently trapped on the DNA strand-block the progression of replication and transcription machineries and hence hamper the faithful transfer of genetic information. However, the repair mechanism of DPCs remains largely elusive. Here we have analyzed the roles of nucleotide excision repair (NER) and homologous recombination (HR) in the repair of DPCs both in vitro and in vivo using a bacterial system. Several lines of biochemical and genetic evidence show that both NER and HR commit to the repair or tolerance of DPCs, but differentially. NER repairs DPCs with crosslinked proteins of sizes less than 12-14 kDa, whereas oversized DPCs are processed exclusively by RecBCD-dependent HR. These results highlight how NER and HR are coordinated when cells need to deal with unusually bulky DNA lesions such as DPCs.

Most common point mutations occurring spontaneously or induced by ionizing radiation are C-->T transitions implicating cytosine as the target. Oxidative cytosine derivatives are the most abundant and mutagenic DNA damage induced by oxidative stress. Base excision repair (BER) pathway initiated by DNA glycosylases is thought to be the major pathway for the removal of these lesions. However, in alternative nucleotide incision repair (NIR) pathway the apurinic/apyrimidinic (AP) endonucleases incise DNA duplex 5' to an oxidatively damaged base in a DNA glycosylase-independent manner. Here, we characterized the substrate specificity of human major AP endonuclease, Ape1, towards 5-hydroxy-2'-deoxycytidine (5ohC) and alpha-anomeric 2'-deoxycytidine (alphadC) residues. The apparent kinetic parameters of the reactions suggest that Ape1 and the DNA glycosylases/AP lyases, hNth1 and hNeil1 repair 5ohC with a low efficiency. Nevertheless, due to the extremely high cellular concentration of Ape1, NIR was the major activity towards 5ohC in cell-free extracts. To address the physiological role of NIR function, we have characterized naturally occurring Ape1 variants including amino acids substitutions (E126A, E126D and D148E) and N-terminal truncated forms (NDelta31, NDelta35 and NDelta61). As expected, all Ape1 mutants had proficient AP endonuclease activity, however, truncated forms showed reduced NIR and 3'-->5' exonuclease activities indicating that these two functions are genetically linked and governed by the same amino acid residues. Furthermore, both Ape1-catalyzed NIR and 3'-->5' exonuclease activities generate a single-strand gap at the 5' side of a damaged base but not at an AP site in duplex DNA. We hypothesized that biochemical coupling of the nucleotide incision and exonuclease degradation may serve to remove clustered DNA damage. Our data suggest that NIR is a backup system for the BER pathway to remove oxidative damage to cytosines in vivo.

DNA-protein cross-link (DPC) damage is produced by varieties of genotoxic agents such as ionizing radiation, UV light, aldehydes, and some chemotherapeutic compounds. DPCs would be harmful to cells since proteins immobilized on DNA strands could hamper DNA transactions in cells. However, it has not been clarified how DPCs are repaired in cells. In the present study, we prepared DNA substrates containing defined DPCs and tested them for repair enzymes. We also examined the sensitivity of Escherichia coli to DPC-inducing agents.

Schizosaccharomyces pombe Nthpl, an ortholog of the endonuclease III family, is the sole bifunctional DNA glycosylase encoded in its genome. The enzyme removes oxidative pyrimidine and incises 3' to the apurinic/apyrimidinic (AP) site, leaving 3'-alpha,beta-unsaturated aldehyde. Analysis of nth1 cDNA revealed an intronless structure including 5'- and 3'-untranslated regions. An Nth1p-green fluorescent fusion protein was predominantly localized in the nuclei of yeast cells, indicating a nuclear function. Deletion of nth1 confirmed that Nth1p is responsible for the majority of activity for thymine glycol and AP site incision in the absence of metal ions, while nth1 mutants exhibit hypersensitivity to methylmethanesulfonate (MMS). Complementation of sensitivity by heterologous expression of various DNA glycosylases showed that the methyl-formamidopyrimidine (me-fapy) and/or AP sites are plausible substrates for Nth1p in repairing MMS damage. Apn2p, the major AP endonuclease in S. pombe, also greatly contributes to the repair of MMS damage. Deletion of nth1 from an apn2 mutant resulted in tolerance to MMS damage, indicating that Nth1p-induced 3'-blocks are responsible for MMS sensitivity in apn2 mutants. Overexpression of Apn2p in nth1 mutants failed to suppress MMS sensitivity. These results indicate that Nth1p, not Apn2p, primarily incises AP sites and that the resultant 3'-blocks are removed by the 3'-phosphodiesterase activity of Apn2p. Nth1p is dispensable for cell survival against low levels of oxidative stress, but wild-type yeast became more sensitive than the nth1 mutant at high levels. Overexpression of Nth1p in heavily damaged cells probably induced cell death via the formation of 3'-blocked single-strand breaks.

Nitric oxide (NO) induces deamination of guanine, yielding xanthine and oxanine (Oxa). Furthermore, Oxa reacts with polyamines and DNA binding proteins to form cross-link adducts. Thus, it is of interest how these lesions are processed by DNA repair enzymes in view of the genotoxic mechanism of NO. In the present study, we have examined the repair capacity for Oxa and Oxa-spermine cross-link adducts (Oxa-Sp) of enzymes involved in base excision repair (BER) and nucleotide excision repair (NER) to delineate the repair mechanism of nitrosative damage to guanine. Oligonucleotide substrates containing Oxa and Oxa-Sp were incubated with purified BER and NER enzymes or cell-free extracts (CFEs), and the damage-excising or DNA-incising activity was compared with that for control (physiological) substrates. The Oxa-excising activities of Escherichia coli and human DNA glycosylases and HeLa CFEs were 0.2-9% relative to control substrates, implying poor processing of Oxa by BER. In contrast, DNA containing Oxa-Sp was incised efficiently by UvrABC nuclease and SOS-induced E.coli CFEs, suggesting a role of NER in ameliorating genotoxic effects associated with nitrosative stress. Analyses of the activity of CFEs from NER-proficient and NER-deficient human cells on Oxa-Sp DNA confirmed further the involvement of NER in the repair of nitrosative DNA damage.

Nitric oxide and nitrous acid induce deamination of DNA bases, resulting in uracil, hypoxanthine, xanthine, and oxanine (Oxa) as major damage. Oxa reacts further with polyamines and DNA binding proteins, generating bulky cross-link adducts. Recently we have shown Oxa and cross-link adducts are potentially genotoxic lesions. In the present study, we have assessed the role of base excision repair (BER) and nucleotide excision repair (NER) systems in the repair of Oxa and Oxa-spermine (Oxa-Sp) cross-link adducts. Oxa was very poorly removed from DNA by both BER glycosylases and NER enzymes, whereas Oxa-Sp was efficiently excised by E. coli and human NER enzymes.

Alpha-anomeric 2'-deoxynucleosides (alphadN) are one of the products formed by ionizing radiation (IR) in DNA under anoxic conditions. Alpha-2'-deoxyadenosine (alphadA) and alpha-thymidine (alphaT) are not recognized by DNA glycosylases, and are likely removed by the alternative nucleotide incision repair (NIR) pathway. Indeed, it has been shown that alphadA is a substrate for the Escherichia coli Nfo and human Ape1 proteins. However, the repair pathway for removal of alphadA and other alphadN in yeast is unknown. Here we report that alphadA when present in DNA is recognized by the Saccharomyces cerevisiae Apn1 protein, a homologue of Nfo. Furthermore, alphaT is a substrate for Nfo and Apn1. Kinetic constants indicate that alphadA and alphaT are equally good substrates, as a tetrahydrofuranyl (THF) residue, for Nfo and Apn1. Using E. coli and S. cerevisiae cell-free extracts, we have further substantiated the role of the nfo and apn1 gene products in the repair of alphadN. Surprisingly, we found that bacteria and yeast NIR-deficient mutants are not sensitive to IR, suggesting that DNA strand breaks with terminal 3'-blocking groups rather than alphadN might contribute to cell survival. We propose that the novel substrate specificities of Nfo and Apn1 play an important role in counteracting oxidative DNA base damage.

Clustered DNA damage (locally multiply damaged site) is thought to be a critical lesion caused by ionizing radiation, and high LET radiation such as heavy ion particles is believed to produce high yields of such damage. Since heavy ion particles are major components of ionizing radiation in a space environment, it is important to clarify the chemical nature and biological consequences of clustered DNA damage and its relationship to the health effects of exposure to high LET particles in humans. The concept of clustered DNA damage emerged around 1980, but only recently has become the subject of experimental studies. In this article, we review methods used to detect clustered DNA damage, and the current status of our understanding of the chemical nature and repair of clustered DNA damage.

Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), previously thought to be a backup enzyme for uracil-DNA glycosylase, has recently been shown to excise 5-hydroxyuracil (hoU), 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU) bearing an oxidized group at ring C5 as well as an uracil. In the present study, we used site-directed mutagenesis to construct a series of mutants of human SMUG1 (hSMUG1), and tested their activity for uracil, hoU, hmU, fU and other bases to elucidate the catalytic and damage-recognition mechanism of hSMUG1. The functional analysis of the mutants, together with the homology modeling of the hSMUG1 structure based on that determined recently for Xenopus laevis SMUG1, revealed the crucial residues for the rupture of the N-glycosidic bond (Asn85 and His239), discrimination of pyrimidine rings through pi-pi stacking to the base (Phe98) and specific hydrogen bonds to the Watson-Crick face of the base (Asn163) and exquisite recognition of the C5 substituent through water-bridged (uracil) or direct (hoU, hmU and fU) hydrogen bonds (Gly87-Met91). Integration of the present results and the structural data elucidates how hSMUG1 accepts uracil, hoU, hmU and fU as substrates, but not other oxidized pyrimidines such as 5-hydroxycytosine, 5-formylcytosine and thymine glycol, and intact pyrimidines such as thymine and cytosine.

Reactive oxygen species from endogenous and environmental sources induce oxidative damage to DNA, and hence pose an enormous threat to the genetic integrity of cells. Such oxidative DNA damage is restored by the base excision repair (BER) pathway that is conserved from bacteria to humans and is initiated by DNA glycosylases, which simply remove the aberrant base from the DNA backbone by hydrolyzing the N-glycosidic bond (monofunctional DNA glycosylase), or further catalyze the incision of a resulting abasic site (bifunctional DNA glycosylase). In human cells, oxidative pyrimidine lesions are generally removed by hNTH1, hNEIL1, or hNEIL2, whereas oxidative purine lesions are removed by hOGG1. hSMUG1 excises a subset of oxidative base damage that is poorly recognized by the above enzymes. Unlike these enzymes, hMYH removes intact A misincorporated opposite template 8-oxoguanine during DNA replication. Although hNTH1, hOGG1, and hMYH account for major cellular glycosylase activity for inherent substrate lesions, mouse models deficient in the enzymes exhibit no overt phenotypes such as the development of cancer, implying backup mechanisms. Contrary to the mouse model, hMYH mutations have been shown to lead to a multiple colorectal adenoma syndrome and high colorectal cancer risk. For cleavage of the N-glycosidic bond, bifunctional DNA glycosylases (hNTH1, hNEIL1, hNEIL2, and hOGG1) use Lys or Pro for direct attack on sugar C1', whereas monofunctional DNA glycosylases (hSMUG1 and hMYH) use an activated water molecule. DNA glycosylases for oxidative damage, if not all, are covalently trapped by DNA containing 2-deoxyribonolactone or oxanine. Thus, the depletion of functional DNA glycosylases using covalent trapping may reduce the BER capacity of cancer cells, hence potentiating the efficacy of anticancer drugs or radiation therapy.

In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5'-dangling modified nucleotide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage- specific endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2'-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2'-deoxyuridine, alpha-2'-deoxyadenosine and alpha-thymidine adducts, generating 3'-hydroxyl and 5'-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly efficient. The substrate specificity and protein conformation of Ape1 is modulated by MgCl2 concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed.

In the accompanying paper [Matsubara, M., et al. (2003) Biochemistry 42, 4993-5002], we have partially purified and characterized rat 5-formyluracil (fU)-DNA glycosylase (FDG). Several lines of evidence have indicated that FDG is a rat homologue of single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1). We report here that rat and human SMUG1 (rSMUG1 and hSMUG1) expressed from the corresponding cDNAs indeed excise fU in single-stranded (ss) and double-stranded (ds) DNA. The enzymes also excised uracil (U) and uracil derivatives bearing an oxidized group at C5 [5-hydroxyuracil (hoU) and 5-hydroxymethyluracil (hmU)] in ssDNA and dsDNA but not analogous cytosine derivatives (5-hydroxycytosine and 5-formylcytosine) and other oxidized damage. The damage specificity and the salt concentration dependence of rSMUG1 (and hSMUG1) agreed well with those of FDG, confirming that FDG is rSMUG1. Consistent with the damage specificity above, hSMUG1 removed damaged bases from Fenton-oxidized calf thymus DNA, generating abasic sites. The amount of resulting abasic sites was about 10% of that generated by endonuclease III or 8-oxoguanine glycosylase in the same substrate. The HeLa cell extract and hSMUG1 exhibited a similar damage preference (hoU.G > hmU.A, fU.A), and the activities for fU, hmU, and hoU in the cell extract were effectively neutralized with hSMUG1 antibodies. These data indicate a dual role of hSMUG1 as a backup enzyme for UNG and a primary repair enzyme for a subset of oxidized pyrimidines such as fU, hmU, and hoU.

Single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1) was previously identified as a putative backup enzyme of major mammalian uracil-DNA glycosylase (UDG). However, the subsequent studies have shown conflicting results about the substrate specificity of SMUG1. In the present study, to clarify the repair role of SMUG1, we determined the damage specificity of purified human SMUG1 (hSMUG1) and its contribution to repair of oxidized bases in HeLa cell extracts.

5-Hydroxyuracil (HOU) and 5-hydroxycytosine (HOC) are major oxidative lesions of cytosine with mutagenic potentials. Therefore, HOU and HOC need to be removed from DNA to avoid mutation. In this study, oligonucleotide substrates containing HOU and HOC were synthesized by DNA polymerase reactions and tested for DNA glycosylases. Ung exhibited an extremely low activity for HOU as compared to uracil (U). In contrast, hSMUG1 excised HOU and U with a comparable efficiency. Ung and hSMUG1 did not excise HOC.

Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions. In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa. The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA [in combination with endonuclease (Endo) IV] and Endo VIII recognized Xan in the tested enzymes. The activity (V(max)/K(m)) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol. The activity of AlkA and Endo VIII for Xan was further substantiated by the release of [(3)H]Xan from the substrate. The treatment of E.coli with N-methyl-N'-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA(+) but not alkA(-) strains. The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain. AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan.

Endonuclease III, encoded by nth in Escherichia coli, removes thymine glycols (Tg), a toxic oxidative DNA lesion. To determine the biological significance of this repair in mammals, we established a mouse model with mutated mNth1, a homolog of nth, by gene targeting. The homozygous mNth1 mutant mice showed no detectable phenotypical abnormality. Embryonic cells with or without wild-type mNth1 showed no difference in sensitivity to menadione or hydrogen peroxide. Tg produced in the mutant mouse liver DNA by X-ray irradiation disappeared with time, though more slowly than in the wild-type mouse. In extracts from mutant mouse liver, we found, instead of mNTH1 activity, at least two novel DNA glycosylase activities against Tg. One activity is significantly higher in the mutant than in wild-type mouse in mitochondria, while the other is another nuclear glycosylase for Tg. These results underscore the importance of base excision repair of Tg both in the nuclei and mitochondria in mammals.

5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species. In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential. Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3'-5'-exonuclease. Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion. However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently. No incorporation of dCMP and dTMP was observed. When fU was substituted for T in templates, the incorporation efficiency of dAMP (f(A) = V(max)/K(m)) decreased to (1/4) to (1/2), depending on the nearest neighbor base pair, and that of dGMP (f(G)) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (f(G)/f(A) for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pK(a) = 8.6) but not T (pK(a) = 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G. The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold). These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic.

Escherichia coli endonuclease III (Endo III) and its eukaryotic homologues are major repair enzymes for pyrimidine lesions formed by reactive oxygen species and ionizing radiation. In the present study, the activities of Endo III and its mouse homologue (mNTH1) have been compared using defined oligonucleotide substrates containing a urea residue (UR), two cis-thymine glycol (TG) diastereoisomers, 5, 6-dihydrothymine (DHT), and 5-hydroxyuracil (HOU). The substrates were incubated with Endo III and mNTH1, and their activities were compared based on the product analysis by gel electrophoresis. Endo III recognized all base lesions tested, but the activity for DHT was extremely lower than other substrates. In contrast, albeit some preference of UR, mNTH1 showed essentially comparable activities for all substrates including DHT. Comparison of the enzymatic parameters for cis-TG and DHT revealed that large decreases in the affinity (K(m), 27-fold) and k(cat) (11-fold) relative to cis-TG made DHT an very poor substrate for Endo III. mNTH1 had comparable affinities and k(cat) for both cis-TG and DHT, though turnover (k(cat)) of mNTH1 was notably slower than Endo III. In view of the reaction mechanism, the paired base effect on the damage recognition by the two enzymes was also examined. The activities of Endo III for UR and HOU were paired base-independent, but those for cis-TG and DHT were significantly enhanced when paired with G. With mNTH1, the paired base effect was evident only for DHT. The variations of the repair activity with paired bases and enzymes are discussed in relation to the base flipping mechanism suggested for base excision repair enzymes.

Rubrobacter radiotolerans is an extremely radioresistant bacterium. It exhibits higher resistance than the well-known radioresistant bacterium Deinococcus radiodurans, but the molecular mechanisms responsible for the radio-resistance of R. radiotolerans remain unknown. In the present study, we have demonstrated the presence of a novel DNA repair enzyme in R. radiotolerans cells that recognizes radiation-induced DNA damages such as thymine glycol, urea residues, and abasic sites. The enzyme was purified from the crude cell extract by a series of chromatography to an apparent physical homogeneity. The purified enzyme showed a single band with a molecular mass of approximately 40 kDa in SDS-polyacrylamide gel electrophoresis, and was designated as R-endonuclease. R-Endonuclease exhibited repair activity for thymine glycol, urea residues, and abasic sites present in plasmid DNA, but did not act on intact DNA, UV-irradiated DNA and DNA containing reduced abasic sites. The substrate specificity together with the salt and pH optima suggests that R-endonuclease is a functional homolog of endonuclease III of Escherichia coli.

7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy) are major DNA lesions formed by reactive oxygen species and are involved in mutagenic and/or lethal events in cells. Both lesions are repaired by human 7, 8-dihydro-8-oxoguanine DNA glycosylase (hOGG1) and formamidopyrimidine DNA glycosylase (Fpg) in human and Escherichia coli cells, respectively. In the present study, the repair activities of hOGG1 and Fpg were compared using defined oligonucleotides containing 8-oxoG and a methylated analog of Fapy (me-Fapy) at the same site. The k(cat)/K(m) values of hOGG1 for 8-oxoG and me-Fapy were comparable, and this was also the case for Fpg. However, the k(cat)/K(m) values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg. Analysis of the Schiff base intermediate by NaBH(4) trapping implied that lower substrate affinity and slower hydrolysis of the intermediate for hOGG1 than Fpg accounted for the difference. hOGG1 and Fpg showed distinct preferences of the base opposite 8-oxoG, with the activity differences being 19.8- (hOGG1) and 12-fold (Fpg) between the most and least preferred bases. Surprisingly, such preferences were almost abolished and less than 2-fold for both enzymes when me-Fapy was a substrate, suggesting that, unlike 8-oxoG, me-Fapy is not subjected to paired base-dependent repair. The repair efficiency of me-Fapy randomly incorporated in M13 DNA varied at the sequence level, but orders of preferred and unpreferred repair sites were quite different for hOGG1 and Fpg. The distinctive activities of hOGG1 and Fpg including enzymatic parameters (k(cat)/K(m)), paired base, and sequence context effects may originate from the differences in the inherent architecture of the DNA binding domain and catalytic mechanism of the enzymes.

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form. In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A. The repair activity for fU was induced in Escherichia coli upon exposure to N-methyl-N'-nitro-N-nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity. Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5-hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate. (1)H and (13)C NMR chemical shifts of 5-formyl-2'-deoxyuridine indicated that the 5-formyl group caused base C-6 and sugar C-1' to be electron deficient, which was shown to result in destabilization of the N-glycosidic bond. These features are common in other good substrates for AlkA and are suggested to play key roles in the differential recognition of fU, hU, and intact thymine. Three mammalian repair enzymes for alkylated and oxidized bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein. In the accompanying paper (Terato, H., Masaoka, A., Kobayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H., J. Biol. Chem. 274, 25144-25150), possible repair mechanisms for fU mispaired with G are reported.

5-Formyluracil (fU), a major methyl oxidation product of thymine, forms correct (fU:A) and incorrect (fU:G) base pairs during DNA replication. In the accompanying paper (Masaoka, A., Terato, H., Kobayashi, M., Honsho, A., Ohyama, Y., and Ide, H. (1999) J. Biol. Chem. 274, 25136-25143), it has been shown that fU correctly paired with A is recognized by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II). In the present work, mispairing frequency of fU with G and cellular repair protein that specifically recognized fU:G mispairs were studied using defined oligonucleotide substrates. Mispairing frequency of fU was determined by incorporation of 2'-deoxyribonucleoside 5'-triphosphate of fU opposite template G using DNA polymerase I Klenow fragment deficient in 3'-5' exonuclease. Mispairing frequency of fU was dependent on the nearest neighbor base pair in the primer terminus and 2-12 times higher than that of thymine at pH 7.8 and 2.6-6.7 times higher at pH 9.0 with an exception of the nearest neighbor T(template):A(primer). AlkA catalyzed the excision of fU placed opposite G, as well as A, and the excision efficiencies of fU for fU:G and fU:A pairs were comparable. In addition, MutS protein involved in methyl-directed mismatch repair also recognized fU:G mispairs and bound them with an efficiency comparable to T:G mispairs, but it did not recognize fU:A pairs. Prior complex formation between MutS and a heteroduplex containing an fU:G mispair inhibited the activity of AlkA to fU. These results suggest that fU present in DNA can be restored by two independent repair pathways, i.e. the base excision repair pathway initiated by AlkA and the methyl-directed mismatch repair pathway initiated by MutS. Biological relevance of the present results is discussed in light of DNA replication and repair in cells.

5-Formyluracil (fU) is an oxidative DNA base damage. This damage has been suggested to be mutagenic and but enzymatic repair of the damage is little known. In this study, repair enzymes that recognize fU have been studied. Kinetic analysis of the repair activity of E. coli 3-methyladenine DNA glycosylase II (AlkA) showed that fU was removed by AlkA with the efficiency comparable to 7-methylguanine. We also examined the participation of the methyl-directed mismatch repair system. The affinity of MutS to the fU:G mispair was essentially similar to that of the T:G mispair that was most efficiently recognized by the MutSLH system. These results suggest two distinct repair pathways of fU in E. coli.

Human methylpurine N-glycosylase (MPG) activity was investigated by monitoring abasic (AP) sites resulting from removal of alkylated bases. The amount of AP sites in MMS-treated HeLa cells transiently increased at 3 h, then gradually decreased to 40% at 24 h. The presence of adenine, an inhibitor of AP endonucleases, in the repair incubation of MMS-treated cells induced moderate accumulation of AP sites, suggesting inhibition of the activities of MPG as well as AP endonucleases by adenine metabolites.

Oligonucleotides containing a unique alpha-deoxyadenosine or tetrahydrofuran (a model abasic site) were synthesized using phosphoramidite chemistry. Repair enzymes from Escherichia coli, including endonucleases III, IV, and VIII, exonuclease III, formamidopyrimidine N-glycosylase, and deoxyinosine 3'-endonuclease, as well as UV dimer N-glycosylases from T4 (den V) and Micrococcus luteus, were examined for their ability to recognize alpha-deoxyadenosine and tetrahydrofuran. In agreement with prior studies, a tetrahydrofuran-containing oligonucleotide was a substrate for endonuclease IV and exonuclease III, but not for the other repair enzymes. However, an oligonucleotide containing alpha-deoxyadenine was a substrate only for endonuclease IV. Competitive inhibition studies with both substrates confirmed that the activity recognizing alpha-deoxyadenine was endonuclease IV and not a possible contaminant in the endonuclease IV preparation. Using E. coli extracts, the activity that recognized alpha-deoxyadenine was dependent on nfo, the structural gene of endonuclease IV, further substantiating that endonuclease IV is the enzyme that recognized alpha-deoxyadenine. Kinetic measurements indicated that alpha-deoxyadenosine was as good a substrate for endonuclease IV as tetrahydrofuran; the Km and Vmax values for both substrates were similar. Using substrates that were labeled at either the 3'- or 5'-terminus, endonuclease IV was shown to hydrolyze the phosphodiester bond 5' to either alpha-deoxyadenosine or tetrahydrofuran, leaving the lesion, alpha-deoxyadenosine or tetrahydrofuran, on the 5'-terminus of the nicked site. The ability of endonuclease IV to recognize alpha-deoxyadenosine suggests that endonuclease IV is able to recognize a new class of DNA base lesions that is not recognized by other DNA N-glycosylases and AP endonucleases.

The processing of unrepaired DNA lesions is a key to understanding and predicting the biological end points of particular DNA damages. In this study, we prepared single-stranded f1 phage (f1-K12) DNA containing dihydrothymine or beta-ureidoisobutyric acid as models for instructive or noninstructive base lesions and assessed the potential biological consequences of these lesions in vitro and in vivo. To determine the effect of the two lesions on in vitro DNA synthesis, the extent of DNA synthesis was measured by 3H-labeled nucleotide incorporation, and the newly synthesized DNA was analyzed by DNA sequencing gels. The results showed that dihydrothymine in the template was at most a weak block to in vitro DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment (Pol I) and T4 DNA polymerase. In contrast, beta-ureidoisobutyric acid constituted a very strong (probably absolute) replicative block in vitro. With Pol I, termination bands were observed either opposite or one base prior to (3' to) the putative beta-ureidoisobutyric acid depending on its position in the template. However, when DNA synthesis was catalyzed by Pol I lacking a 3'----5' exonuclease activity, termination bands were only observed opposite beta-ureidoisobutyric acid, with purine nucleotides being incorporated preferentially opposite the lesion. With T4 DNA polymerase that contains a very active 3'----5' exonuclease activity, DNA synthesis was arrested almost exclusively one base prior to (3' to) the putative beta-ureidoisobutyric acid site in the template. We also measured survival of transfecting DNA containing dihydrothymine or beta-ureidoisobutyric acid in an attempt to correlate the in vitro data with in vivo processing. In keeping with the results obtained in vitro, dihydrothymine present in transfecting f1-K12 DNA did not constitute an inactivating lesion. On the other hand, it took 0.9 beta-ureidoisobutyric acid residues per molecule to inactivate transfecting f1-K12 DNA, indicating that the lesion was an absolute replicative block in vivo. When host cells were ultraviolet-irradiated to induce the SOS response, a slight increase (about 2-fold) in survival of transfecting f1-K12 DNA containing beta-ureidoisobutyric acid was observed. The potential effects of the structures of base lesions on lesion-polymerase interactions are discussed.