DNA repair in response to anthracycline-DNA adducts: a role for both homologous recombination and nucleotide excision repair

Doxorubicin, a widely used anthracycline anticancer agent, acts as a topoisomerase II poison but can also form formaldehyde-mediated DNA adducts. This has led to the development of doxorubicin derivatives such as doxoform, which can readily form adducts with DNA. This work aimed to determine which DNA repair pathways are involved in the recognition and possible repair of anthracycline-DNA adducts. Cell lines lacking functional proteins involved in each of the five main repair pathways, mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end-joining (NHEJ) were examined for sensitivity to various anthracycline adduct-forming treatments. The treatments used were doxorubicin, barminomycin (a model adduct-forming anthracycline) and doxoform (a doxorubicin-formaldehyde conjugate). Cells with deficiencies in MMR, BER and NHEJ were equally sensitive to adduct-forming treatments compared to wild type cells and therefore these pathways are unlikely to play a role in the repair of these adducts. Some cells with deficiencies in the NER pathway (specifically, those lacking functional XPB, XPD and XPG), displayed tolerance to adducts induced by both barminomycin and doxoform and also exhibited a decreased level of apoptosis in response to adduct-forming treatments. Conversely, two HR deficient cell lines were shown to be more sensitive to barminomycin and doxoform than HR proficient cells, indicating that this pathway is also involved in the repair response to anthracycline-DNA adducts. These results suggest an unusual damage response pathway to anthracycline adducts involving both NER and HR that could be used to optimise cancer therapy for tumours with either high levels of NER or defective HR. Tumours with either of these characteristics would be predicted to respond particularly well to anthracycline-DNA adduct-forming treatments.     

Genotoxicity and mutagenicity of chromium(VI)/ascorbate-generated DNA adducts in human and bacterial cells

Reduction of carcinogenic Cr(VI) by vitamin C generates ascorbate-Cr(III)-DNA cross-links, binary Cr(III)-DNA adducts, and can potentially cause oxidative DNA damage by intermediate reaction products. Here, we examined the mutational spectrum and the importance of different forms of DNA damage in genotoxicity and mutagenicity of Cr(VI) activated by physiological concentrations of ascorbate. Reduction of Cr(VI) led to a dose-dependent formation of both mutagenic and replication-blocking DNA lesions as detected by propagation of the pSP189 plasmids in human fibroblasts. Disruption of Cr-DNA binding abolished mutagenic responses and normalized the yield of replicated plasmids, indicating that Cr-DNA adducts were responsible for both mutagenicity and genotoxicity of Cr(VI). The absence of DNA breaks and abasic sites confirmed the lack of a significant production of hydroxyl radicals and Cr(V)-peroxo complexes in Cr(VI)-ascorbate reactions. Ascorbate-Cr(III)-DNA cross-links were much more mutagenic than smaller Cr(III)-DNA adducts and accounted for more than 90% of Cr(VI) mutagenicity. Ternary adducts were also several times more potent in the inhibition of replication than binary complexes. The Cr(VI)-induced mutational spectrum consisted of an approximately equal number of deletions and G/C-targeted point mutations (51% G/C --> T/A and 30% G/C --> A/T). In Escherichia coli cells, Cr(VI)-induced DNA adducts were only highly genotoxic but not mutagenic under either normal or SOS-induced conditions. Lower toxicity and high mutagenicity of ascorbate-Cr(III)-DNA adducts in human cells may result from the recruitment of an error-prone bypass DNA polymerase(s) to the stalled replication forks. Our results suggest that phosphotriester-type DNA adducts could play a more important role in human than bacterial mutagenesis.     

Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase eta

Platinum anticancer agents form bulky DNA adducts which are thought to exert their cytotoxic effect by blocking DNA replication. Translesion synthesis, one of the pathways of postreplication repair, is thought to account for some resistance to DNA damage and much of the mutagenicity of bulky DNA adducts in dividing cells. Oxaliplatin has been shown to be effective in cisplatin-resistant cell lines and less mutagenic than cisplatin in the Ames assay. We have shown that the eukaryotic DNA polymerases yeast pol zeta, human pol beta, and human pol gamma bypass oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. Human pol eta, a product of the XPV gene, has been shown to catalyze efficient translesion synthesis past cis, syn-cyclobutane pyrimidine dimers. In the present study we compared translesion synthesis past different Pt-GG adducts by human pol eta. Our data show that, similar to other eukaryotic DNA polymerases, pol eta bypasses oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. However, pol eta-catalyzed translesion replication past Pt-DNA adducts was more efficient and less accurate than that seen for previously tested polymerases. We show that the efficiency and fidelity of translesion replication past Pt-DNA adducts appear to be determined by both the structure of the adduct and the DNA polymerase active site.     

DNA错配修复与癌症的发生及治疗

DNA错配修复是细胞复制后的一种修复机制,具有维持DNA复制保真度,控制基因变异的作用。DNA错配修复缺陷使整个基因组不稳定,最终会导致肿瘤和癌症的发生。DNA错配修复系统不仅通过矫正在DNA重组和复制过程中产生的碱基错配而保持基因组的稳定,而且通过诱导DNA损伤细胞的凋亡而消除由突变细胞生长形成的癌变。错配修复缺陷细胞的抗药性也引起了癌症化疗研究方面的关注。大多数情况下,错配修复健全型细胞对肿瘤化疗药物敏感,而错配修复缺陷细胞却有较高的抗性。DNA错配修复系统通过修复和诱导细胞凋亡维护基因组稳定的功能,显示了错配修复途径在癌症生物学和分子医学中的重要性。     

Both replication bypass fidelity and repair efficiency influence the yield of mutations per target dose in intact mammalian cells induced by benzo[a]pyrene-diol-epoxide and dibenzo[a,l]pyrene-diol-epoxide

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

Mismatch repair and DNA damage signalling

Postreplicative mismatch repair (MMR) increases the fidelity of DNA replication by up to three orders of magnitude, through correcting DNA polymerase errors that escaped proofreading. MMR also controls homologous recombination (HR) by aborting strand exchange between divergent DNA sequences. In recent years, MMR has also been implicated in the response of mammalian cells to DNA damaging agents. Thus, MMR-deficient cells were shown to be around 100-fold more resistant to killing by methylating agents of the S(N)1type than cells with functional MMR. In the case of cisplatin, the sensitivity difference was lower, typically two- to three-fold, but was observed in all matched MMR-proficient and -deficient cell pairs. More controversial is the role of MMR in cellular response to other DNA damaging agents, such as ionizing radiation (IR), topoisomerase poisons, antimetabolites, UV radiation and DNA intercalators. The MMR-dependent DNA damage signalling pathways activated by the above agents are also ill-defined. To date, signalling cascades involving the Ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), as well as the stress-activated kinases JNK/SAPK and p38alpha have been linked with methylating agent and 6-thioguanine (TG) treatments, while cisplatin damage was reported to activate the c-Abl and JNK/SAPK kinases in MMR-dependent manner. MMR defects are found in several different cancer types, both familiar and sporadic, and it is possible that the involvement of the MMR system in DNA damage signalling play an important role in transformation. The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.     

Microsatellite instability and suppressed DNA repair enzyme expression in rheumatoid arthritis

Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can potentially induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity during DNA replication. Key members of the MMR system include MutSalpha (hMSH2 and hMSH6) and MutSbeta (hMSH2 and hMSH3). To provide evidence of DNA damage in inflamed synovium, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells of RA patients using specific primer sequences for five key microsatellites. Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis tissue. Western blot analysis for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the NO donor S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated constitutive expression of hMSH2, 3, and 6 in RA and osteoarthritis FLS. When FLS were cultured with S-nitroso-N-acetylpenicillamine, the pattern of MMR expression in RA synovium was reproduced (high hMSH3, low hMSH6). Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.     

The Escherichia coli methyl-directed mismatch repair system repairs base pairs containing oxidative lesions

A major role of the methyl-directed mismatch repair (MMR) system of Escherichia coli is to repair postreplicative errors. In this report, we provide evidence that MMR also acts on oxidized DNA, preventing mutagenesis. When cells deficient in MMR are grown anaerobically, spontaneous mutation frequencies are reduced compared with those of the same cells grown aerobically. In addition, we show that a dam mutant has an increased sensitivity to hydrogen peroxide treatment that can be suppressed by mutations that inactivate MMR. In a dam mutant, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted to single- and double-strand DNA breaks. Thus, base pairs containing oxidized bases will be converted to strand breaks if they are repaired by MMR. This is demonstrated by the increased peroxide sensitivity of a dam mutant and the finding that the sensitivity can be suppressed by mutations inactivating MMR. We demonstrate further that this repair activity results from MMR recognition of base pairs containing 8-oxoguanine (8-oxoG) based on the finding that overexpression of the MutM oxidative repair protein, which repairs 8-oxoG, can suppress the mutH-dependent increase in transversion mutations. These findings demonstrate that MMR has the ability to prevent oxidative mutagenesis either by removing 8-oxoG directly or by removing adenine misincorporated opposite 8-oxoG or both.     

Human Pluripotent Stem Cells Have a Novel Mismatch Repair-dependent Damage Response

Human pluripotent stem cells (PSCs) are presumed to have robust DNA repair pathways to ensure genome stability. PSCs likely need to protect against mutations that would otherwise be propagated throughout all tissues of the developing embryo. How these cells respond to genotoxic stress has only recently begun to be investigated. Although PSCs appear to respond to certain forms of damage more efficiently than somatic cells, some DNA damage response pathways such as the replication stress response may be lacking. Not all DNA repair pathways, including the DNA mismatch repair (MMR) pathway, have been well characterized in PSCs to date. MMR maintains genomic stability by repairing DNA polymerase errors. MMR is also involved in the induction of cell cycle arrest and apoptosis in response to certain exogenous DNA-damaging agents. Here, we examined MMR function in PSCs. We have demonstrated that PSCs contain a robust MMR pathway and are highly sensitive to DNA alkylation damage in an MMR-dependent manner. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cell types. In somatic cells, a permanent G₂/M cell cycle arrest is induced in the second cell cycle after DNA damage. The PSCs, however, directly undergo apoptosis in the first cell cycle. This response reveals that PSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation. Our results also suggest an alternative molecular mechanism by which the MMR pathway can induce a response to DNA damage that may have implications for tumorigenesis.     

UV- and MMS-induced mutagenesis of lambdaO(am)8 phage under nonpermissive conditions for phage DNA replication

Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant.     

Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli.

Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.     

Mismatch repair causes the dynamic release of an essential DNA polymerase from the replication fork

Mismatch repair (MMR) corrects DNA polymerase errors occurring during genome replication. MMR is critical for genome maintenance, and its loss increases mutation rates several hundred fold. Recent work has shown that the interaction between the mismatch recognition protein MutS and the replication processivity clamp is important for MMR in Bacillus subtilis. To further understand how MMR is coupled to DNA replication, we examined the subcellular localization of MMR and DNA replication proteins fused to green fluorescent protein (GFP) in live cells, following an increase in DNA replication errors. We demonstrate that foci of the essential DNA polymerase DnaE-GFP decrease following mismatch incorporation and that loss of DnaE-GFP foci requires MutS. Furthermore, we show that MutS and MutL bind DnaE in vitro, suggesting that DnaE is coupled to repair. We also found that DnaE-GFP foci decrease in vivo following a DNA damage-independent arrest of DNA synthesis showing that loss of DnaE-GFP foci is caused by perturbations to DNA replication. We propose that MutS directly contacts the DNA replication machinery, causing a dynamic change in the organization of DnaE at the replication fork during MMR. Our results establish a striking and intimate connection between MMR and the replicating DNA polymerase complex in vivo.     

Endogenous formation and significance of 1,N2-propanodeoxyguanosine adducts

The detection of 1,N2-propanodeoxyguanosine adducts in the DNA of rodent and human tissues as endogenous lesions has raised important questions regarding the source of their formation and their roles in carcinogenesis. Both in vitro and in vivo studies have generated substantial evidence which supports the involvement of short- and long-chain enals derived from oxidized polyunsaturated fatty acids (PUFAs) in their formation. These studies show that: (1) the cyclic propano adducts are common products from reactions of enals with DNA bases; (2) they are formed specifically from linoleic acid (LA; omega-6) and docosahexaenoic acid (omega-3) under in vitro stimulated lipid peroxidation conditions; (3) the levels of propano adducts are dramatically increased in rat liver DNA upon depletion of glutathione; (4) the adduct levels are increased in the liver DNA of the CCl4-treated rats and the mutant strain of Long Evans rats which are genetically predisposed to increased lipid peroxidation; and (5) adduct levels are significantly higher in older rats than in newborn rats. These studies collectively demonstrate that tissue lipid peroxidation is a main endogenous pathway leading to propano adduction in DNA. The possible contribution from environmental sources, however, cannot be completely excluded. The mutagenicity of enals and the mutations observed in site-specific mutagenesis studies using a model 1,N2-propanodeoxyguanosine adduct suggest that these adducts are potential promutagenic lesions. The increased levels of the propano adducts in the tissue of carcinogen-treated animals also provide suggestive evidence for their roles in carcinogenesis. The involvement of these adducts in tumor promotion is speculated on the basis that oxidative condition in tissues is believed to be associated with this process.     

Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells

DNA interstrand crosslinks (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. ICLs are induced in DNA by intercalating compounds such as the widely used therapeutic agent psoralen. In bacteria, both nucleotide excision repair (NER) and homologous recombination are required for the repair of ICLs. The processing of ICLs in mammalian cells is not clearly understood. However, it is known that processing can occur by NER, which for psoralen ICLs can be an error-generating process conducive to mutagenesis. We show here that another repair pathway, mismatch repair (MMR), is also involved in eliminating psoralen ICLs in human cells. MMR deficiency renders cells hypersensitive to psoralen ICLs without diminishing their mutagenic potential, suggesting that MMR does not contribute to error-generating repair, and that MMR may represent a relatively error-free mechanism for processing these lesions in human cells. Thus, enhancement of MMR relative to NER may reduce the mutagenesis caused by DNA ICLs in humans.     

Specificity of replicative and SOS-inducible DNA polymerases in frameshift mutagenesis: mutability of Salmonella typhimurium strains overexpressing SOS-inducible DNA polymerases to 30 chemical mutagens

DNA replication is frequently hindered because of the presence of DNA lesions induced by endogenous and exogenous genotoxic agents. To circumvent the replication block, cells are endowed with multiple specialized DNA polymerases that can bypass a variety of DNA damage. To better understand the specificity of specialized DNA polymerases to bypass lesions, we have constructed a set of derivatives of Salmonella typhimurium TA1538 harboring plasmids carrying the polB, dinB or mucAB genes encoding Escherichia coli DNA polymerase II, DNA polymerase IV or DNA polymerase RI, respectively, and examined the mutability to 30 chemicals. The parent strain TA1538 possesses CGCGCGCG hotspot sequence for -2 frameshift. Interestingly, the chemicals could be classified into four groups based on the mutagenicity to the derivatives: group I whose mutagenicity was highest in strain YG5161 harboring plasmid carrying dinB; group II whose mutagenicity was almost equally high in strain YG5161 and strain TA98 harboring plasmid carrying mucAB; group III whose mutagenicity was highest in strain TA98; group IV whose mutagenicity was not affected by the introduction of any of the plasmids. Introduction of plasmid carrying polB did not enhance the mutagenicity except for benz[a]anthracene. We also introduced a plasmid carrying polA encoding E. coli DNA polymerase I to strain TA1538. Strikingly, the introduction of the plasmid reduced the mutagenicity of chemicals belonging to groups I, II and III, but not the chemicals of group IV, to the levels observed in the derivative whose SOS-inducible DNA polymerases were all deleted. These results suggest that (i) DNA polymerase IV and DNA polymerase RI possess distinct but partly overlapping specificity to bypass lesions leading to -2 frameshift, (ii) the replicative DNA polymerase, i.e., DNA polymerase III, participates in the mutagenesis and (iii) the enhanced expression of E. coli polA may suppress the access of Y-family DNA polymerases to the replication complex.     

Pol kappa partially rescues MMR-dependent cytotoxicity of O6-methylguanine

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine ( O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding.     

Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.     

Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair

Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10⁵], and this precision is improved to about [1/10⁷] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (G^O) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, G^O/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of G^O might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR.     

Relationship between mutagenicity and DNA adduct formation in mammalian cells for fjord- and bay-region diol-epoxides of polycyclic aromatic hydrocarbons

Chinese hamster V79 cells were treated with the anti- and syn-diastereomers of the bay- or fjord-region diol-epoxides of four polycyclic aromatic hydrocarbons, namely benzo[a]pyrene (BP), benzo[c]chrysene (BcC), benzo[g]chrysene (BgC) and benzo[c]phenanthrene (BcPh). The frequency of induction of 6-thioguanine-resistant mutations was determined, and the extent of formation of DNA adducts was measured by 32P-postlabelling. When expressed as mutation frequency per nanomoles compound per millilitre incubation medium, this group of chemicals expressed a 160-fold range in potency. In agreement with previous experimental studies, the anti-diol-epoxide of BcC was highly mutagenic, inducing in excess of 3 x 10(4) mutations/10(6) cells per nmol compound/ml. The mutagenic activities of the anti- and syn-diol-epoxides of BP were 10- and 100-fold lower, respectively. Both diol-epoxides of BgC, the syn-BcC and the anti-BcPh derivatives were also highly mutagenic, and only the syn-BcPh diol-epoxide was less mutagenic than the anti-diol-epoxide of BP. Determination of the levels of DNA adducts formed by the diol-epoxides indicated that the most mutagenic compounds were the most DNA reactive, although the fjord-region diol-epoxides gave rise to more complex patterns of adducts than those of the BP diol-epoxides. When the mutagenicity results were expressed as mutations per femtomoles total adducts formed, all compounds showed similar activities. Thus the potent mutagenicity of the fjord region diol-epoxides appears to be due to the high frequency with which they form DNA adducts in V79 cells, rather than to formation of adducts with greater mutagenic potential.