Disparate contributions of the Fanconi anemia pathway and homologous recombination in preventing spontaneous mutagenesis

Fanconi anemia (FA) is a chromosomal instability disorder in which DNA-damage processing defects are reported for translesion synthesis (TLS), non-homologous end joining (NHEJ) and homologous recombination (HR; both increased and decreased). To reconcile these diverse findings, we compared spontaneous mutagenesis in FA and HR mutants of hamster CHO cells. In the fancg mutant we find a reduced mutation rate accompanied by an increased proportion of deletions within the hprt gene. Moreover, in fancg cells gene amplification at the CAD and dhfr loci is elevated, another manifestation of inappropriate processing of damage during DNA replication. In contrast, the rad51d HR mutant has a greatly elevated rate of hprt mutations, >85% of which are deletions. Our analysis supports the concept that HR faithfully restores broken replication forks, whereas the FA pathway acts more globally to ensure chromosome stability by promoting efficient end joining of replication-derived breaks, as well as TLS and HR.     

SCAI promotes error‐free repair of DNA interstrand crosslinks via the Fanconi anemia pathway

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI‐FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)‐mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error‐free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error‐free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI‐deficient cells are exquisitely sensitive to ICL‐inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR‐mediated ICL repair is defective, and breaks are instead re‐ligated by polymerase θ‐dependent microhomology‐mediated end‐joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error‐free ICL repair.     

C. elegans FANCD2 responds to replication stress and functions in interstrand cross-link repair

One of the least well understood DNA repair processes in cells is the repair of DNA interstrand cross-links (ICLs) which present a major obstacle to DNA replication and must be repaired or bypassed to allow fork progression. Fanconi anemia (FA) is an inherited genome instability syndrome characterized by hypersensitivity to ICL damage. Central to the FA repair pathway is FANCD2 that is mono-ubiquitylated in response to replication stress and ICL damage through the action of the FA core complex and its E3-ubiquitin ligase subunit, FANCL. In its mono-ubiquitylated form FANCD2 is recruited to repair foci where it is believed to somehow coordinate ICL repair and restart of impeded replication forks. However, the precise mechanism through which the FA pathway and mono-ubiquitylation of FANCD2 promotes ICL repair remains unclear. Here we report on a functional homologue of FANCD2 in C. elegans (FCD-2). Although fcd-2 mutants are homozygous viable, they are exquisitely sensitive to ICL-inducing agents, but insensitive to ionizing radiation (IR). fcd-2 is dispensable for meiotic recombination and activation of the S-phase checkpoint, indicating that ICL sensitivity is likely due to a repair rather than a signalling defect. Indeed, we show that FCD-2 is mono-ubiquitylated in response to ICL damage and is recruited to nuclear repair foci. Consistent with the sensitivity of fcd-2 mutants, FCD-2 focus formation is induced in response to ICL damage and replication stress, but not following IR, suggesting that FCD-2 responds to lesions that block DNA replication and not DNA double strand breaks per se. The realization that the FA pathway is conserved in a genetically tractable model system will permit the comprehensive analysis of the interplay between the FA, homologous recombination (HR), translesion synthesis (TLS) and nucleotide excision repair (NER) pathways, critical to the understanding of ICL repair.     

Involvement of mouse Rev3 in tolerance of endogenous and exogenous DNA damage

The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta that is implicated in mutagenic translesion synthesis of damaged DNA. To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3. Although some strain-dependent variation was observed, Rev3(-/-) embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities. Rev3(-/-) cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability. Histochemical analysis of Rev3(-/-) embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages. Although increased levels of p53 are detected in Rev3(-/-) embryos, the embryonic phenotype was not rescued by the absence of p53. A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3(-/-) embryos. The inner cell mass of cultured Rev3(-/-) blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene. These combined data are compatible with a model in which, in the absence of polymerase zeta, double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response. Together, these data are consistent with involvement of polymerase zeta in translesion synthesis of endogenously and exogenously induced DNA lesions.     

The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair

The Fanconi anemia (FA) protein FANCC is essential for chromosome stability in vertebrate cells, a feature underscored by the extreme sensitivity of FANCC-deficient cells to agents that crosslink DNA. However, it is not known how this FA protein facilitates the repair of both endogenously acquired and mutagen-induced DNA damage. Here, we use the model vertebrate cell line DT40 to address this question. We discover that apart from functioning in homologous recombination, FANCC also promotes the mutational repair of endogenously generated abasic sites. Moreover in these vertebrate cells, the efficient repair of crosslinks requires the combined functions of FANCC, translesion synthesis, and homologous recombination. These studies reveal that the FA proteins cooperate with key mutagenesis and repair processes that enable replication of damaged DNA.     

Nucleotide excision repair or polymerase V-mediated lesion bypass can act to restore UV-arrested replication forks in Escherichia coli

Nucleotide excision repair and translesion DNA synthesis are two processes that operate at arrested replication forks to reduce the frequency of recombination and promote cell survival following UV-induced DNA damage. While nucleotide excision repair is generally considered to be error free, translesion synthesis can result in mutations, making it important to identify the order and conditions that determine when each process is recruited to the arrested fork. We show here that at early times following UV irradiation, the recovery of DNA synthesis occurs through nucleotide excision repair of the lesion. In the absence of repair or when the repair capacity of the cell has been exceeded, translesion synthesis by polymerase V (Pol V) allows DNA synthesis to resume and is required to protect the arrested replication fork from degradation. Pol II and Pol IV do not contribute detectably to survival, mutagenesis, or restoration of DNA synthesis, suggesting that, in vivo, these polymerases are not functionally redundant with Pol V at UV-induced lesions. We discuss a model in which cells first use DNA repair to process replication-arresting UV lesions before resorting to mutagenic pathways such as translesion DNA synthesis to bypass these impediments to replication progression.     

The Fanconi anemia core complex is required for efficient point mutagenesis and Rev1 foci assembly

Fanconi anemia (FA) is a chromosome instability syndrome characterized by congenital abnormalities, cellular hypersensitivity to DNA crosslinking agents, and heightened cancer risk. Eight of the thirteen identified FA genes encode subunits of a nuclear FA core complex that monoubiquitinates FANCD2 and FANCI to maintain genomic stability in response to replication stress. The FA pathway has been implicated in the regulation of error-prone DNA damage tolerance via an undefined molecular mechanism. Here, we show that the FA core complex is required for efficient spontaneous and UVC-induced point mutagenesis, independently of FANCD2 and FANCI. Consistent with the observed hypomutability of cells deficient in the FA core complex, we also demonstrate that these cells are impaired in the assembly of the error-prone translesion DNA synthesis polymerase Rev1 into nuclear foci. Consistent with a role downstream of the FA core complex and like known FA proteins, Rev1 is required to prevent DNA crosslinker-induced chromosomal aberrations in human cells. Interestingly, proliferating cell nuclear antigen (PCNA) monoubiquitination, known to contribute to Rev1 recruitment, does not require FA core complex function. Our results suggest a role for the FA core complex in regulating Rev1-dependent DNA damage tolerance independently of FANCD2, FANCI, and PCNA monoubiquitination.     

Asymmetry of DNA replication and translesion synthesis of UV-induced thymine dimers

In vitro replication assays for detection and quantification of bypass of UV-induced DNA photoproducts were used to compare the capacity of extracts prepared from different human cell lines to replicate past the cis,syn cyclobutane thymine dimer ([c,s]TT). The results demonstrated that neither nucleotide excision repair (NER) nor mismatch repair (MMR) activities in the intact cells interfered with measurements of bypass replication efficiencies in vitro. Extracts prepared from HeLa (NER- and MMR-proficient), xeroderma pigmentosum group A (NER-deficient), and HCT116 (MMR-deficient) cells displayed similar capacity for translesion synthesis, when the substrate carried the site-specific [c,s]TT on the template for the leading or the lagging strand of nascent DNA. Extracts from xeroderma pigmentosum variant cells, which lack DNA polymerase eta, were devoid of bypass activity. Bypass-proficient extracts as a group (n=16 for 3 extracts) displayed higher efficiency (P=0.005) for replication past the [c,s]TT during leading strand synthesis (84+/-22%) than during lagging strand synthesis (64+/-13%). These findings are compared to previous results concerning the bypass of the (6-4) photoproduct [Biochemistry 40 (2001) 15215] and analyzed in the context of the reported characteristics of bypass DNA polymerases implicated in translesion synthesis of UV-induced DNA lesions. Models to explain how these enzymes might interact with the DNA replication machinery are considered. An alternative pathway of bypass replication, which avoids translesion synthesis, and the mutagenic potential of post-replication repair mechanisms that contribute to the duplication of the human genome damaged by UV are discussed.     

Mechanisms and consequences of methylating agent-induced SCEs and chromosomal aberrations: a long road traveled and still a far way to go

Since the milestone work of Evans and Scott, demonstrating the replication dependence of alkylation-induced aberrations, and Obe and Natarajan, pointing to the critical role of DNA double-strand breaks (DSBs) as the ultimate trigger of aberrations, the field has grown extensively. A notable example is the identification of DNA methylation lesions provoking chromosome breakage (clastogenic) effects, which made it possible to model clastogenic pathways evoked by genotoxins. Experiments with repair-deficient mutants and transgenic cell lines revealed both O6-methylguanine (O6MeG) and N- methylpurines as critical lesions. For S(N)2 agents such as methyl- methanesulfonate (MMS), base N-methylation lesions are most critical, likely because of the formation of apurinic sites blocking replication. For S(N)1 agents, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O6-methylguanine (O6MeG) plays the major role both in recombination and clastogenicity in the post-treatment cell cycle, provided the lesion is not pre-replicatively repaired by O6-methylguanine-DNA methyltransferase (MGMT). The conversion probability of O6MeG into SCEs and chromosomal aberrations is estimated to be about 30:1 and >10,000:1 respectively, indicating this mispairing pro-mutagenic lesion to be highly potent in inducing recombination giving rise to SCEs. O6MeG needs replication and mismatch repair to become converted into a critical secondary genotoxic lesion. Here it is proposed that this secondary lesion can be tolerated by a process termed recombination bypass. This process is supposed to be important in the tolerance of lesions that can not be processed by translesion synthesis accomplished by low-fidelity DNA polymerases. Recombination bypass results in SCEs and might represent an alternative pathway of tolerance of non-instructive lesions. In the case of O6MeG-derived secondary lesions, recombination bypass appears to protect against cell killing since SCEs are already induced with low, non-toxic doses of MNNG. Saturation of lesion tolerance by recombination bypass or translesion synthesis may cause block of DNA replication leading to DSBs at stalled replication forks, which result in chromatid-type aberrations. Along with this model, several putative consequences of methylation-induced aberrations will be discussed such as cell death by apoptosis as well its role in tumor promotion and progression.     

Tolerating DNA damage during eukaryotic chromosome replication

In eukaryotes, the evolutionarily conserved RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired DNA lesions that interfere with the progression of replication forks, helping to ensure the completion of chromosome replication and the maintenance of genome stability in every cell cycle. This pathway uses two different strategies for damage bypass: translesion DNA synthesis, which is carried out by specialized polymerases that can replicate across the lesions, and DNA damage avoidance, a process that relies on a switch to an undamaged-DNA template for synthesis past the lesion. In this review, we summarise the current knowledge on DNA damage tolerance mechanisms mediated by RAD6/RAD18 that are used by eukaryotic cells to cope with DNA lesions during chromosome replication.     

The p-benzoquinone DNA adducts derived from benzene are highly mutagenic

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

Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines

The ability to bypass DNA lesions encountered during replication is important in order to maintain cell viability and avoid genomic instability. Exposure of mammalian cells to UV-irradiation induces the formation of DNA lesions that stall replication forks. In order to restore replication, different bypass mechanisms are operating, previously named post-replication repair. Translesion DNA synthesis is performed by low-fidelity polymerases, which can replicate across damaged sites. The nature of lesions and of polymerases involved influences the resulting frequency of mutations. Homologous recombination represents an alternative pathway for the rescue of stalled replication forks. Caffeine has long been recognized to influence post-replication repair, although the mechanism is not identified. Here, we found that caffeine delays the progress of replication forks in UV-irradiated Chinese hamster cells. The length of this enhanced delay was similar in wild-type cells and in cell deficient in either homologous recombination or nucleotide excision repair. Furthermore, caffeine attenuated the frequency of UV-induced mutations in the hprt gene, whereas the frequency of recombination, monitored in this same gene, was enhanced. These observations indicate that in cells exposed to UV-light, caffeine inhibits the rescue of stalled replication forks by translesion DNA synthesis, thereby causing a switch to bypass via homologous recombination. The biological consequence of the former pathway is mutations, while the latter results in chromosomal aberrations.     

Arrest of S-phase progression is impaired in Fanconi anemia cells

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability, hypersensitivity to DNA cross-linking agents, and a prolonged G2 phase of the cell cycle. We observed a marked dose-dependent accumulation of FA cells in the G2 compartment after treatment with 4,5',8-trimethylpsoralen (Me(3)Pso) in combination with 365 nm irradiation. Using bivariate DNA distribution methodology, we determined the proportion of replicating and arresting S-phase cells and observed that, whereas normal cells arrested DNA replication in the presence of Me(3)Pso cross-links and monoadducts, FA lymphoblasts failed to arrest DNA synthesis. Taken together, the above data suggest that, in response to damage induced by DNA cross-linking agents, the S-phase checkpoint is inefficient in FA cells. This would lead to accumulation of secondary lesions, such as single- and double-strand breaks and gaps. The prolonged time in G2 phase seen in FA cells therefore exists in order to allow the cells to remove lesions which accumulated during the preceding abnormal S phase.     

DNA sequence analysis of 1-nitropyrene-4,5-oxide and 1-nitropyrene-9,10-oxide induced mutations in the hprt gene of Chinese hamster ovary cells

Nitropyrene, the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagenic and tumorigenic environmental pollutant that requires metabolic activation via nitroreduction and ring oxidation. In order to determine the role of ring oxidation in the mutagenicity of 1-nitropyrene, its oxidative metabolites, 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, were synthesized and their mutation spectra were determined in the coding region of hprt gene of CHO cells by a PCR amplification of reverse-transcribed hprt mRNA, followed by a DNA sequence analysis. A comparison of the two metabolites for mutation frequencies showed that 1-nitropyrene 9,10-oxide was 2-times higher than 1-nitropyrene 4,5-oxide. The mutation spectrum for 1-nitropyrene 4,5-oxide was base substitutions (33/49), one base deletions (11/49) and exon deletions (5/49). In the case of 1-nitropyrene 9,10-oxide, base substitutions (27/50), one base deletions (15/50), and exon deletions (8/50) were observed. Base substitutions were distributed randomly throughout the hprt gene. The majority of the base substitutions in mutant from 1-nitropyrene 4,5-oxide treated cells were A-->G transition (15/33) and G-->A transition (8/33). The predominant base substitution, A-->G transition (11/27) and G-->A transition (8/27), were also observed in mutant from 1-nitropyrene 9,10-oxide treated cells. The mutation at the site of adenine and guanine was consistent with the previous results, where the sites of DNA adduct formed by these compounds were predominant at the sites of purines. A comparison of the mutational patterns between 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide showed that there were no significant differences in the overall mutational spectrum. These results indicate that each oxidative metabolite exhibits an equal contribution to the mutagenicity of 1-nitropyrene, and ring oxidation of 1-nitropyrene is an important metabolic pathway to the formation of significant lethal DNA lesions.     

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.     

Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells

Glycidamide (GA)-induced mutagenesis in mammalian cells is not very well understood. Here, we investigated mutagenicity and DNA repair of GA-induced adducts utilizing Chinese hamster cell lines deficient in base excision repair (BER), nucleotide excision repair (NER) or homologous recombination (HR) in comparison to parent wild-type cells. We used the DRAG assay in order to map pathways involved in the repair of GA-induced DNA lesions. This assay utilizes the principle that a DNA repair deficient cell line is expected to be affected in growth and/or survival more than a repair proficient cell. A significant induction of mutations by GA was detected in the hprt locus of wild-type cells but not in BER deficient cells. Cells deficient in HR or BER were three or five times, respectively, more sensitive to GA in terms of growth inhibition than were wild-type cells. The results obtained on the rate of incisions in BER and NER suggest that lesions induced by GA are repaired by short patch BER rather than long patch BER or NER. Furthermore, a large proportion of the GA-induced lesions gave rise to strand breaks that are repaired by a mechanism not involving PARP. It is suggested that these strand breaks, which might be the results from alkylation of the backbone phosphate, are misrepaired by HR during replication thereby leading to a clastogenic rather than a mutagenic pathway. The type of lesion responsible for the mutagenic effect of GA cannot be concluded from the results presented in this study.