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The regulation of replication is essential to preserve genome integrity. Mms1 is part of the E3 ubiquitin ligase complex that is linked to replication fork progression. By identifying Mms1 binding sites genome-wide in Saccharomyces cerevisiae we connected Mms1 function to genome integrity and replication fork progression at particular G-rich motifs. This motif can form G-quadruplex (G4) structures in vitro. G4 are stable DNA structures that are known to impede replication fork progression. In the absence of Mms1, genome stability is at risk at these G-rich/G4 regions as demonstrated by gross chromosomal rearrangement assays. Mms1 binds throughout the cell cycle to these G-rich/G4 regions and supports the binding of Pif1 DNA helicase. Based on these data we propose a mechanistic model in which Mms1 binds to specific G-rich/G4 motif located on the lagging strand template for DNA replication and supports Pif1 function, DNA replication and genome integrity.
© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
To protect cells from oxidative DNA damage and mutagenesis, organisms possess multiple glycosylases to recognize the damaged bases and to initiate the Base Excision Repair pathway. Three DNA glycosylases have been identified in mammals that are homologous to the Escherichia coli Fpg and Nei proteins, Neil1, Neil2, and Neil3. Neil1 and Neil2 in human and mouse have been well characterized while the properties of the Neil3 protein remain to be elucidated. In this study, we report the characterization of Mus musculus (house mouse) Neil3 (MmuNeil3) as an active DNA glycosylase both in vitro and in vivo. In duplex DNA, MmuNeil3 recognizes the oxidized purines, spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino- 5-formamidopyrimidine (FapyA), but not 8-oxo-7,8-dihydroguanine (8-oxoG). Interestingly, MmuNeil3 prefers lesions in single-stranded DNA and in bubble structures. In contrast to other members of the family that use the N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine. We expressed the glycosylase domain of MmuNeil3 (MmuNeil3Delta324) in an Escherichia coli triple mutant lacking Fpg, Nei, and MutY glycosylase activities and showed that MmuNeil3 greatly reduced both the spontaneous mutation frequency and the level of FapyG in the DNA, suggesting that Neil3 plays a role in repairing FapyG in vivo.
The 5R thymine glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5'-GTgG-3' sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson-Crick alignment. The Tg N3H imine and A N(6) amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G*C base pair is disrupted. The solvent accessible surface and T(2) relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH(3) group is favored; propeller twisting of the Tg*A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3'-neighbor guanine alleviate steric clash with the 5'-neighbor base pair. Tg also destabilizes the 5'-neighbor G*C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA.
Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.
Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3' incision reaction. Here we report the crystal structure of the N-terminal catalytic domain of UvrC at 1.5 A resolution, which catalyzes the 3' incision reaction and shares homology with the catalytic domain of the GIY-YIG family of intron-encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium-water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY-YIG endonuclease domains. Structural and biochemical data strongly suggest that the N-terminal endonuclease domain of UvrC utilizes a novel one-metal mechanism to cleave the phosphodiester bond.
The dihalomethane CH(2)Cl(2) is an industrial solvent of potential concern to humans because of its potential genotoxicity and carcinogenicity. To characterize DNA damage by dihalomethanes, a rapid DNA digestion under acidic conditions was developed to identify alkali labile DNA-dihalomethane nucleoside adducts using HPLC-electrospray mass spectrometry. DNA digestion worked best using pH 5.0 sodium acetate buffer, a 30 min incubation with DNase II and phosphodiesterase II, and a 2 h acid phosphatase digest. DNA was modified with S-(1-acetoxymethyl)glutathione (GSCH(2)OAc), a reagent modeling activated dihalomethanes. Adducts to G, A, and T were detected at high ratios of GSCH(2)OAc/DNA following digestion of the DNA with the procedure used here. The relative efficacy of adduct formation was G > T > A > C. The four DNA nucleosides were also reacted with the dihalomethanes CH(2)Cl(2) and CH(2)Br(2) in the presence of glutathione (GSH) and GSH S-transferases from bacteria (DM11), rat (GST 5-5), and human (GST T1-1) under conditions that produce mutations in bacteria. All enzymes formed adducts to all four nucleosides, with dGuo being the most readily modified nucleoside. Thus, the pattern paralleled the results obtained with the model compounds GSCH(2)OAc and DNA. CH(2)Cl(2) and CH(2)Br(2) yielded similar amounts of adducts under these conditions. The relative efficiency of adduct formation by GSH transferases was rat 5-5 > human T1-1 > bacterial DM11, showing that human GSH transferase T1-1 can form dihalomethane adducts under the conditions used. Although the lability of DNA adducts has precluded more sophisticated experiments and in vivo studies have not yet been possible, the work collectively demonstrates the ability of several GSH transferases to generate DNA adducts from dihalomethanes, with G being the preferred site of adduction in both this and the GSCH(2)OAc model system.
Endonuclease G (EndoG) is a nuclear-encoded mitochondrial protein reported to be important for both nuclear DNA fragmentation during apoptosis and mitochondrial DNA replication. To evaluate the in vivo function of EndoG, we have investigated the effects of EndoG deficiency in cells and mice. We found that EndoG homozygous mutant embryos die between embryonic days 2.5 and 3.5. Mitochondrial DNA copy numbers in ovulated oocytes from EndoG heterozygous mutant and wild-type mice are similar, suggesting that EndoG is involved in a cellular function unrelated to mitochondrial DNA replication. Interestingly, we found that cells from EndoG heterozygous mutant mice exhibit increased resistance to both tumor necrosis factor alpha- and staurosporine-induced cell death. Moreover, spontaneous cell death of spermatogonia in EndoG heterozygous mutant mice is significantly reduced compared with wild-type mice. DNA fragmentation is also reduced in EndoG+/- thymocytes and splenocytes compared with wild-type cells, as well as in EndoG+/- thymus in vivo compared with that of the wild-type mice, on activation of apoptosis. These findings indicate that EndoG is essential during early embryogenesis and plays a critical role in normal apoptosis and nuclear DNA fragmentation.
Mus81, a fission yeast protein related to the XPF subunit of ERCC1-XPF nucleotide excision repair endonuclease, is essential for meiosis and important for coping with stalled replication forks. These processes require resolution of X-shaped DNA structures known as Holliday junctions. We report that Mus81 and an associated protein Eme1 are components of an endonuclease that resolves Holliday junctions into linear duplex products. Mus81 and Eme1 are required during meiosis at a late step of meiotic recombination. The mus81 meiotic defect is rescued by expression of a bacterial Holliday junction resolvase. These findings constitute strong evidence that Mus81 and Eme1 are subunits of a nuclear Holliday junction resolvase.
S-(1-Acetoxymethyl)glutathione (GSCH(2)OAc) was synthesized and used as a model for the reaction of glutathione (GSH)-dihaloalkane conjugates with nucleosides and DNA. Previously, S-[1-(N(2)-deoxyguanosinyl)methyl]GSH had been identified as the major adduct formed in the reaction of GSCH(2)OAc with deoxyguanosine. GSCH(2)OAc was incubated with the three remaining deoxyribonucleosides to identify other possible adducts. Adducts to all three nucleosides were found using electrospray ionization mass spectrometry (ESI MS). The adduct of GSCH(2)OAc and deoxyadenosine was formed in yield of up to 0.05% and was identified as S-[1-(N(7)-deoxyadenosinyl)methyl]GSH. The pyrimidine deoxyribonucleoside adducts were formed more efficiently, resulting in yields of 1 and 2% for the GSCH(2)OAc adducts derived from thymidine and deoxycytidine, respectively, but their lability prevented their structural identification by (1)H NMR. On the basis of the available UV spectra, we propose the structures S-[1-(N(3)-thymidinyl)methyl]GSH and S-[1-(N(4)-deoxycytidinyl)methyl]GSH. Because adduct degradation occurred most rapidly at alkaline and neutral pH values, an enzymatic DNA digestion procedure was developed for the rapid hydrolysis of DNA to deoxyribonucleosides at acidic pH. DNA digests were completed in less than 2 h with a two-step method, which consisted of a 15 min incubation of DNA with high concentrations of deoxyribonuclease II and phosphodiesterase II at pH 4.5, followed by incubation of resulting nucleotides with acid phosphatase. Analysis of the hydrolysis products by HPLC-ESI-MS indicated the presence of the thymidine adduct.
We report the identities of the members of a group of proteins that associate with BRCA1 to form a large complex that we have named BASC (BRCA1-associated genome surveillance complex). This complex includes tumor suppressors and DNA damage repair proteins MSH2, MSH6, MLH1, ATM, BLM, and the RAD50-MRE11-NBS1 protein complex. In addition, DNA replication factor C (RFC), a protein complex that facilitates the loading of PCNA onto DNA, is also part of BASC. We find that BRCA1, the BLM helicase, and the RAD50-MRE11-NBS1 complex colocalize to large nuclear foci that contain PCNA when cells are treated with agents that interfere with DNA synthesis. The association of BRCA1 with MSH2 and MSH6, which are required for transcription-coupled repair, provides a possible explanation for the role of BRCA1 in this pathway. Strikingly, all members of this complex have roles in recognition of abnormal DNA structures or damaged DNA, suggesting that BASC may serve as a sensor for DNA damage. Several of these proteins also have roles in DNA replication-associated repair. Collectively, these results suggest that BRCA1 may function as a coordinator of multiple activities required for maintenance of genomic integrity during the process of DNA replication and point to a central role for BRCA1 in DNA repair.