Anastasia Tsaalbi-Shtylik

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.

Separate domains of Rev1 mediate two modes of DNA damage bypass in mammalian cells.

ABSTRACT The Y family DNA polymerase Rev1 has been proposed to play a regulatory role in the replication of damaged templates. To elucidate the mechanism by which Rev1 promotes DNA damage bypass, we have analyzed the progression of replication on UV light-damaged DNA in mouse embryonic fibroblasts that contain a defined deletion in the N-terminal BRCT domain of Rev1 or that are deficient for Rev1. We provide evidence that Rev1 plays a coordinating role in two modes of DNA damage bypass, i.e., an early and a late pathway. The cells carrying the deletion in the BRCT domain are deficient for the early pathway, reflecting a role of the BRCT domain of Rev1 in mutagenic translesion synthesis. Rev1-deficient cells display a defect in both modes of DNA damage bypass. Despite the persistent defect in the late replicational bypass of fork-blocking (6-4)pyrimidine-pyrimidone photoproducts, overall replication is not strongly affected by Rev1 deficiency. This results in almost completely replicated templates that contain gaps encompassing the photoproducts. These gaps are inducers of DNA damage signaling leading to an irreversible G2 arrest. Our results corroborate a model in which Rev1-mediated DNA damage bypass at postreplicative gaps quenches irreversible DNA damage responses.

Mammalian polymerase ζ is essential for post-replication repair of UV-induced DNA lesions

DNA polymerase zeta is believed to be an essential constituent of DNA damage tolerance, comprising several pathways that allow the replication of DNA templates containing unrepaired damage. We wanted to better define the role of polymerase zeta in DNA damage tolerance in mammalian cells. To this aim we have investigated replication of ultraviolet light-damaged DNA templates in mouse embryonic fibroblasts deficient for Rev3, the catalytic subunit of polymerase zeta. We found that Rev3 is important for a post-replication repair pathway of helix-distorting [6-4]pyrimidine-pyrimidone photoproducts and, to a lesser extent, of cyclobutane pyrimidine dimers. Unlike its partner Rev1, Rev3 appears not to be involved in an immediate translesion synthesis pathway at a stalled replication fork. The deficiency of Rev3(-/-) MEFs in post-replication repair of different photoproducts contributes to the extreme sensitivity of these cells to UV light.

The translesion polymerase Rev3L in the tolerance of alkylating anticancer drugs

Temozolomide and fotemustine, representing methylating and chloroethylating agents, respectively, are used in the treatment of glioma and malignant melanoma. Because chemoresistance of these tumors is a common phenomenon, identification of the underlying mechanisms is needed. Here we show that Rev3L, the catalytic subunit of the translesion DNA polymerase ζ, mediates resistance to both temozolomide and fotemustine. Rev3L knockout cells are hypersensitive to both agents. It is remarkable that cells heterozygous for Rev3L showed an intermediate sensitivity. Rev3L is not involved in the tolerance of the toxic O6-methylguanine lesion. However, a possible role of Rev3L in the tolerance of O6-chloroethylguanine or the subsequently formed N1-guanine-N3-cytosine interstrand cross-link is shown. Rev3L had no influence on base excision repair (BER) of the N-alkylation lesions but is very likely to be involved in the tolerance of N-alkylations or apurinic/apyrimidinic sites originating from them. We also show that Rev3L exerts its protective effect in replicating cells and that loss of Rev3L leads to a significant increase in DNA double-strand breaks after temozolomide and fotemustine treatment. These data show that Rev3L contributes to temozolomide and fotemustine resistance, thus acting in concert with O6-methylguanine-DNA methyltransferase, BER, mismatch repair, and double-strand break repair in defense against simple alkylating anticancer drugs.

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.

Rev1 is essential in generating G to C transversions downstream of the Ung2 pathway but not the Msh2+Ung2 hybrid pathway

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes are initiated by the enzymatic deamination of cytosine (C) to uracil (U). Uracil‐DNA‐glycosylase (Ung2) converts uracils into apyrimidinic (AP) sites, which is essential for the generation of transversions (TVs) at G/C basepairs during SHM and for efficient DNA break formation during CSR. Besides Ung2, the mismatch repair protein Msh2 and the translesion synthesis (TLS) DNA polymerase (Pol) Rev1 are implicated in SHM and CSR. To further unravel the role of Rev1, we studied WT, Rev1‐deficient, Msh2‐deficient, and Rev1, Msh2 double‐deficient B cells. Loss of Rev1 only slightly reduced CSR. During SHM G/C to C/G TVs are generated in both Ung2‐ and Ung+Msh2‐dependent fashions. We found that Rev1 is essential for the Msh2‐independent generation of these TVs downstream of Ung2‐induced AP sites. In the Ung+Msh2 hybrid pathway, Rev1 is not essential and can be substituted by an alternative TLS Pol, especially when Rev1 is lacking.

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.

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.

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

An Msh2/Msh6-dependent DNA repair mechanism mitigates the mutagenicity of photolesions and induces cell cycle responses by excising incorrect nucleotides incorporated by postreplicative translesion synthesis.

Abrogation of Microsatellite-instable Tumors Using a Highly Selective Suicide Gene/Prodrug Combination

A substantial fraction of sporadic and inherited colorectal and endometrial cancers in humans is deficient in DNA mismatch repair (MMR). These cancers are characterized by length alterations in ubiquitous simple sequence repeats, a phenotype called microsatellite instability. Here we have exploited this phenotype by developing a novel approach for the highly selective gene therapy of MMR-deficient tumors. To achieve this selectivity, we mutated the VP22FCU1 suicide gene by inserting an out-of-frame microsatellite within its coding region. We show that in a significant fraction of microsatellite-instable (MSI) cells carrying the mutated suicide gene, full-length protein becomes expressed within a few cell doublings, presumably resulting from a reverting frameshift within the inserted microsatellite. Treatment of these cells with the innocuous prodrug 5-fluorocytosine (5-FC) induces strong cytotoxicity and we demonstrate that this owes to multiple bystander effects conferred by the suicide gene/prodrug combination. In a mouse model, MMR-deficient tumors that contained the out-of-frame VP22FCU1 gene displayed strong remission after treatment with 5-FC, without any obvious adverse systemic effects to the mouse. By virtue of its high selectivity and potency, this conditional enzyme/prodrug combination may hold promise for the treatment or prevention of MMR-deficient cancer in humans.