Induction of mismatch repair deficiency, compromised DNA damage signaling and compound hypermutagenesis by a dietary mutagen in a cell-based model for Lynch syndrome.

The prevalent cancer predisposition Lynch syndrome (LS, OMIM #120435) is caused by an inherited heterozygous defect in any of the four core DNA mismatch repair (MMR) genes MSH2, MSH6, MLH1 or PMS2. MMR repairs errors by the replicative DNA polymerases in all proliferating tissues. Its deficiency, following somatic loss of the wild-type copy, results in a spontaneous mutator phenotype that underlies the rapid development of, predominantly, colorectal cancer (CRC) in LS. Here, we have addressed the hypothesis that aberrant responses of intestinal stem cells to diet-derived mutagens may be causally involved in the restricted cancer tropism of LS. To test this we have generated a panel of isogenic mouse embryonic stem (mES) cells with heterozygous or homozygous disruption of multiple MMR genes and investigated their responses to the common dietary mutagen and carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Our data reveal that PhIP can inactivate the wild-type allele of heterozygous mES cells via the induction of either loss of heterozygosity (LOH) or intragenic mutations. Moreover, while protective DNA damage signaling (DDS) is compromised, PhIP induces more mutations in Msh2, Mlh1, Msh6 or Pms2-deficient mES cells than in wild-type cells. Combined with their spontaneous mutator phenotypes, this results in a compound hypermutator phenotype. Together, these results indicate that dietary mutagens may promote CRC development in LS at multiple levels, providing a rationale for dietary modifications in the management of LS.

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage.

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage.

FANCD2 and REV1 cooperate in the protection of nascent DNA strands in response to replication stress.

REV1 is a eukaryotic member of the Y-family of DNA polymerases involved in translesion DNA synthesis and genome mutagenesis. Recently, REV1 is also found to function in homologous recombination. However, it remains unclear how REV1 is recruited to the sites where homologous recombination is processed. Here, we report that loss of mammalian REV1 results in a specific defect in replication-associated gene conversion. We found that REV1 is targeted to laser-induced DNA damage stripes in a manner dependent on its ubiquitin-binding motifs, on RAD18, and on monoubiquitinated FANCD2 (FANCD2-mUb) that associates with REV1. Expression of a FANCD2-Ub chimeric protein in RAD18-depleted cells enhances REV1 assembly at laser-damaged sites, suggesting that FANCD2-mUb functions downstream of RAD18 to recruit REV1 to DNA breaks. Consistent with this suggestion we found that REV1 and FANCD2 are epistatic with respect to sensitivity to the double-strand break-inducer camptothecin. REV1 enrichment at DNA damage stripes also partially depends on BRCA1 and BRCA2, components of the FANCD2/BRCA supercomplex. Intriguingly, analogous to FANCD2-mUb and BRCA1/BRCA2, REV1 plays an unexpected role in protecting nascent replication tracts from degradation by stabilizing RAD51 filaments. Collectively these data suggest that REV1 plays multiple roles at stalled replication forks in response to replication stress.

Persistently stalled replication forks inhibit nucleotide excision repair in trans by sequestering Replication protein A.

Rev3, the catalytic subunit of DNA polymerase ζ, is essential for translesion synthesis of cytotoxic DNA photolesions, whereas the Rev1 protein plays a noncatalytic role in translesion synthesis. Here, we reveal that mammalian Rev3(-/-) and Rev1(-/-) cell lines additionally display a nucleotide excision repair (NER) defect, specifically during S phase. This defect is correlated with the normal recruitment but protracted persistence at DNA damage sites of factors involved in an early stage of NER, while repair synthesis is affected. Remarkably, the NER defect becomes apparent only at 2 h post-irradiation indicating that Rev3 affects repair synthesis only indirectly, rather than performing an enzymatic role in NER. We provide evidence that the NER defect is caused by scarceness of Replication protein A (Rpa) available to NER, resulting from its sequestration at stalled replication forks. Also the induction of replicative stress using hydroxyurea precludes the accumulation of Rpa at photolesion sites, both in Rev3(-/-) and in wild-type cells. These data support a model in which the limited Rpa pool coordinates replicative stress and NER, resulting in increased cytotoxicity of ultraviolet light when replicative stress exceeds a threshold.

Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light.

Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6-4) pyrimidine pyrimidone photoproducts ((6-4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6-4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6-4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects.

Analysis of mutant frequencies and mutation spectra in hMTH1 knockdown TK6 cells exposed to UV radiation.

Ultraviolet radiation is a highly mutagenic agent that damages the DNA by the formation of mutagenic photoproducts at dipyrimidine sites and by oxidative DNA damages via reactive oxygen species (ROS). ROS can also give rise to mutations via oxidation of dNTPs in the nucleotide pool, e.g. 8-oxo-dGTP and 2-OH-dATP and subsequent incorporation during DNA replication. Here we show that expression of human MutT homolog 1 (hMTH1) which sanitizes the nucleotide pool by dephosphorylating oxidized dNTPs, protects against mutagenesis induced by long wave UVA light and by UVB light but not by short wave UVC light. Mutational spectra analyses of UVA-induced mutations at the endogenous Thymidine kinase gene in human lymphoblastoid cells revealed that hMTH1 mainly protects cells from transitions at GC and AT base pairs.

Strand-biased defect in C/G transversions in hypermutating immunoglobulin genes in Rev1-deficient mice.

Somatic hypermutation of Ig genes enables B cells of the germinal center to generate high-affinity immunoglobulin variants. Key intermediates in somatic hypermutation are deoxyuridine lesions, introduced by activation-induced cytidine deaminase. These lesions can be processed further to abasic sites by uracil DNA glycosylase. Mutagenic replication of deoxyuridine, or of its abasic derivative, by translesion synthesis polymerases is hypothesized to underlie somatic hypermutation. Rev1 is a translesion synthesis polymerase that in vitro incorporates uniquely deoxycytidine opposite deoxyuridine and abasic residues. To investigate a role of Rev1 in mammalian somatic hypermutation we have generated mice deficient for Rev1. Although Rev1-/- mice display transient growth retardation, proliferation of Rev1-/- LPS-stimulated B cells is indistinguishable from wild-type cells. In mutated Ig genes from Rev1-/- mice, C to G transversions were virtually absent in the nontranscribed (coding) strand and reduced in the transcribed strand. This defect is associated with an increase of A to T, C to A, and T to C substitutions. These results indicate that Rev1 incorporates deoxycytidine residues, most likely opposite abasic nucleotides, during somatic hypermutation. In addition, loss of Rev1 causes compensatory increase in mutagenesis by other translesion synthesis polymerases.

Error-prone translesion replication of damaged DNA suppresses skin carcinogenesis by controlling inflammatory hyperplasia.

The induction of skin cancer involves both mutagenic and proliferative responses of the epidermis to ultraviolet (UV) light. It is believed that tumor initiation requires the mutagenic replication of damaged DNA by translesion synthesis (TLS) pathways. The mechanistic basis for the induction of proliferation, providing tumor promotion, is poorly understood. Here, we have investigated the role of TLS in the initiation and promotion of skin carcinogenesis, using a sensitive nucleotide excision repair-deficient mouse model that carries a hypomorphic allele of the error-prone TLS gene Rev1. Despite a defect in UV-induced mutagenesis, skin carcinogenesis was accelerated in these mice. This paradoxical phenotype was caused by the induction of inflammatory hyperplasia of the mutant skin that provides strong tumor promotion. The induction of hyperplasia was associated with mild and transient replicational stress of the UV-damaged genome, triggering DNA damage signaling and senescence. The concomitant expression of Interleukin-6 (IL-6) is in agreement with an executive role for IL-6 and possibly other cytokines in the autocrine induction of senescence and the paracrine induction of inflammatory hyperplasia. In conclusion, error-prone TLS suppresses tumor-promoting activities of UV light, thereby controlling skin carcinogenesis.

Differential activity of UV-DDB in mouse keratinocytes and fibroblasts: impact on DNA repair and UV-induced skin cancer.

UV-damaged DNA-binding protein (UV-DDB) is essential for global genome nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers (CPD) and accelerates repair of 6-4 photoproducts (6-4PP). The high UV-induced skin cancer susceptibility of mice compared to man has been attributed to low expression of the UV-DDB subunit DDB2 in mouse skin cells. However, DDB2 knockout mice exhibit enhanced UVB skin carcinogenesis indicating that DDB2 protects mice against UV-induced skin cancer. To resolve these apparent contradictory findings, we systematically investigated the NER capacity of mouse fibroblasts and keratinocytes. Compared to fibroblasts, keratinocytes exhibited an increased level of UV-DDB activity, contained significantly higher levels of other NER proteins (i.e. XPC and XPB) and displayed efficient repair of CPD. At low UVB dosages, the difference in skin cancer susceptibility between DDB2 KO and wild type mice was even much more pronounced than previously reported with high dose UVB exposures. Hence, our observations show that mouse keratinocytes express sufficient levels of UV-DDB for efficient repair of photolesions and efficient protection against UV-induced skin cancer at physiological relevant UV exposure.

DNA mismatch repair-dependent DNA damage responses and cancer.

Canonical DNA mismatch repair (MMR) excises base-base mismatches to increase the fidelity of DNA replication. Thus, loss of MMR leads to increased spontaneous mutagenesis. MMR genes also are involved in the suppression of mutagenic, and the induction of protective, responses to various types of DNA damage. In this review we describe these non-canonical roles of MMR at different lesion types. Loss of non-canonical MMR gene functions may have important ramifications for the prevention, development and treatment of colorectal cancer associated with inherited MMR gene defects in Lynch syndrome. This graphical review pays tribute to Samuel H. Wilson. Sam not only made seminal contributions to understanding base excision repair, particularly with respect to structure-function relationships in DNA polymerase ß but also, as Editor of DNA Repair, has maintained a high standard of the journal.

Gene transcription increases DNA damage-induced mutagenesis in mammalian stem cells.

DNA damage-induced mutations in actively transcribed genes in stem cells underlie genetic diseases including cancer. Here we investigated whether transcription affects DNA damage-induced gene mutations in mouse embryonic stem cells. To this aim we developed cell lines in which transcription of an Hprt minigene reporter, located at a different genomic positions, is regulated by the tTA2 Tetracycline-controlled transactivator. This allows detection of mutagenic events at both Hprt and tTA2 using a single selection. We found that UV-C and benzo[a]pyrenediolepoxide induced significantly more mutations at the Hprt minigene when the gene was transcribed. The transcription-associated increase in UV-C-induced mutagenesis appears independent of the integration site of the Hprt minigene. Molecular analysis of UV-induced Hprt mutants revealed that transcription of damaged DNA enhances the frequency of nucleotide substitutions and triggers the generation of intragenic deletions at the Hprt minigene. We speculate that these deletions are a result of error-prone DNA end-joining of double strand DNA breaks that are generated when replication forks collide with transcription complexes stalled at DNA lesions.

Reduced methylation-induced mutagenesis in rat splenocytes in vivo by sub-chronic low dose exposure to N-metyl-N-nitrosourea.

Estimates of genotoxic effects of mutagens at low and protracted doses are often based on linear extrapolation of data obtained at relatively high doses. To test the validity of such an approach, a comparison was made between the mutagenicity of N-methyl-N-nitrosourea (MNU) in T-lymphocytes of the rat following two treatment protocols, i.e. sub-chronic exposure to a low dose (15-45 repeated exposures to 1mg/kg of MNU) or acute exposure to a single high dose (15, 30 or 45 mg/kg of MNU). Mutation induction appeared dramatically lower following sub-chronic treatment compared to treatment with a single high exposure. Furthermore, DNA sequence analysis of the coding region of the hprt gene in MNU-induced mutants showed that acute high dose treatment causes mainly GC-->AT base pair changes, whereas sub-chronic treatment results in a significant contribution of AT base pair changes to mutation induction. We hypothesize that O(6)-methylguanine-DNA methyltransferase is saturated after acute treatments, while after sub-chronic treatment most O(6)-methylguanine is efficiently repaired. These data suggest (i) that risk estimations at low and protracted doses of MNU on the basis of linear extrapolation of effects measured at high dose are too high and (ii) that the protective effects of DNA repair processes are relatively strong at low sub-chronic exposure.

The BRCT domain of mammalian Rev1 is involved in regulating DNA translesion synthesis.

Rev1 is a deoxycytidyl transferase associated with DNA translesion synthesis (TLS). In addition to its catalytic domain, Rev1 possesses a so-called BRCA1 C-terminal (BRCT) domain. Here, we describe cells and mice containing a targeted deletion of this domain. Rev1(B/B) mice are healthy, fertile and display normal somatic hypermutation. Rev1(B/B) cells display an elevated spontaneous frequency of intragenic deletions at Hprt. In addition, these cells were sensitized to exogenous DNA damages. Ultraviolet-C (UV-C) light induced a delayed progression through late S and G2 phases of the cell cycle and many chromatid aberrations, specifically in a subset of mutant cells, but not enhanced sister chromatid exchanges (SCE). UV-C-induced mutagenesis was reduced and mutations at thymidine-thymidine dimers were absent in Rev1(B/B) cells, the opposite phenotype of UV-C-exposed cells from XP-V patients, lacking TLS polymerase eta. This suggests that the enhanced UV-induced mutagenesis in XP-V patients may depend on error-prone Rev1-dependent TLS. Together, these data indicate a regulatory role of the Rev1 BRCT domain in TLS of a limited spectrum of endogenous and exogenous nucleotide damages during a defined phase of the cell cycle.

Roles of mutagenic translesion synthesis in mammalian genome stability, health and disease.

Most spontaneous and DNA damage-induced nucleotide substitutions in eukaryotes depend on translesion synthesis polymerases Rev1 and Pol ζ, the latter consisting of the catalytic subunit Rev3 and the accessory protein Rev7. Here we review the regulation, and the biochemical and cellular functions, of Rev1/Pol ζ-dependent translesion synthesis. These are correlated with phenotypes of mouse models with defects in Rev1, Rev3 or Rev7. The data indicate that Rev1/Pol ζ-mediated translesion synthesis is important for adaptive immunity while playing paradoxical roles in oncogenesis. On the other hand, by enabling the replication of endogenously damaged templates, Rev1/Pol ζ -dependent translesion synthesis protects stem cells, thereby preventing features of ageing. In conclusion, Rev1/Pol ζ-dependent translesion synthesis at DNA helix-distorting nucleotide lesions orchestrates pleiotropic responses that determine organismal fitness and disease.

Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions.

In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa-Atr-Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects.

De novo mutations in PLXND1 and REV3L cause Möbius syndrome.

Möbius syndrome (MBS) is a neurological disorder that is characterized by paralysis of the facial nerves and variable other congenital anomalies. The aetiology of this syndrome has been enigmatic since the initial descriptions by von Graefe in 1880 and by Möbius in 1888, and it has been debated for decades whether MBS has a genetic or a non-genetic aetiology. Here, we report de novo mutations affecting two genes, PLXND1 and REV3L in MBS patients. PLXND1 and REV3L represent totally unrelated pathways involved in hindbrain development: neural migration and DNA translesion synthesis, essential for the replication of endogenously damaged DNA, respectively. Interestingly, analysis of Plxnd1 and Rev3l mutant mice shows that disruption of these separate pathways converge at the facial branchiomotor nucleus, affecting either motoneuron migration or proliferation. The finding that PLXND1 and REV3L mutations are responsible for a proportion of MBS patients suggests that de novo mutations in other genes might account for other MBS patients.

Temporally distinct translesion synthesis pathways for ultraviolet light-induced photoproducts in the mammalian genome.

Replicative polymerases (Pols) arrest at damaged DNA nucleotides, which induces ubiquitination of the DNA sliding clamp PCNA (PCNA-Ub) and DNA damage signaling. PCNA-Ub is associated with the recruitment or activation of translesion synthesis (TLS) DNA polymerases of the Y family that can bypass the lesions, thereby rescuing replication and preventing replication fork collapse and consequent formation of double-strand DNA breaks. Here, we have used gene-targeted mouse embryonic fibroblasts to perform a comprehensive study of the in vivo roles of PCNA-Ub and of the Y family TLS Pols η, ι, κ, Rev1 and the B family TLS Polζ in TLS and in the suppression of DNA damage signaling and genome instability after exposure to UV light. Our data indicate that TLS Pols ι and κ and the N-terminal BRCT domain of Rev1, that previously was implicated in the regulation of TLS, play minor roles in TLS of DNA photoproducts. PCNA-Ub is critical for an early TLS pathway that replicates both strongly helix-distorting (6-4) pyrimidine-pyrimidone ((6-4)PP) and mildly distorting cyclobutane pyrimidine dimer (CPD) photoproducts. The role of Polη is mainly restricted to early TLS of CPD photoproducts, whereas Rev1 and, in particular, Polζ are essential for the bypass of (6-4)PP photoproducts, both early and late after exposure. Thus, structurally distinct photoproducts at the mammalian genome are bypassed by different TLS Pols in temporally different, PCNA-Ub-dependent and independent fashions.

Different sets of translesion synthesis DNA polymerases protect from genome instability induced by distinct food-derived genotoxins.

DNA lesions, induced by genotoxic compounds, block the processive replication fork but can be bypassed by specialized translesion synthesis (TLS) DNA polymerases (Pols). TLS safeguards the completion of replication, albeit at the expense of nucleotide substitution mutations. We studied the in vivo role of individual TLS Pols in cellular responses to benzo[a]pyrene diolepoxide (BPDE), a polycyclic aromatic hydrocarbon, and 4-hydroxynonenal (4-HNE), a product of lipid peroxidation. To this aim, we used mouse embryonic fibroblasts with targeted disruptions in the TLS-associated Pols η, ι, κ, and Rev1 as well as in Rev3, the catalytic subunit of TLS Polζ. After exposure, cellular survival, replication fork progression, DNA damage responses (DDR), and the induction of micronuclei were investigated. The results demonstrate that Rev1, Rev3, and, to a lesser extent, Polη are involved in TLS and the prevention of DDR and of DNA breaks, in response to both agents. Conversely, Polκ and the N-terminal BRCT domain of Rev1 are specifically involved in TLS of BPDE-induced DNA damage. We furthermore describe a novel role of Polι in TLS of 4-HNE-induced DNA damage in vivo. We hypothesize that different sets of TLS polymerases act on structurally different genotoxic DNA lesions in vivo, thereby suppressing genomic instability associated with cancer. Our experimental approach may provide a significant contribution in delineating the molecular bases of the genotoxicity in vivo of different classes of DNA-damaging agents.

The Rev1 translesion synthesis polymerase has multiple distinct DNA binding modes.

Rev1 is a eukaryotic DNA polymerase of the Y family involved in translesion synthesis (TLS), a major damage tolerance pathway that allows DNA replication at damaged templates. Uniquely amongst the Y family polymerases, the N-terminal part of Rev1, dubbed the BRCA1 C-terminal homology (BRCT) region, includes a BRCT domain. While most BRCT domains mediate protein-protein interactions, Rev1 contains a predicted α-helix N-terminal to the BRCT domain and in human Replication Factor C (RFC) such a BRCT region endows the protein with DNA binding capacity. Here, we studied the DNA binding properties of yeast and mouse Rev1. Our results show that the BRCT region of Rev1 specifically binds to a 5' phosphorylated, recessed, primer-template junction. This DNA binding depends on the extra α-helix, N-terminal to the BRCT domain. Surprisingly, a stretch of 20 amino acids N-terminal to the predicted α-helix is also critical for high-affinity DNA binding. In addition to 5' primer-template junction binding, Rev1 efficiently binds to a recessed 3' primer-template junction. These dual DNA binding characteristics are discussed in view of the proposed recruitment of Rev1 by 5' primer-template junctions, downstream of stalled replication forks.