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 G 2 arrest. Our results corroborate a model in which Rev1-mediated DNA damage bypass at postreplicative gaps quenches irreversible DNA damage responses.Unrepaired DNA damage usually leads to an arrest of replicative polymerases. Nonetheless, prokaryotic and eukaryotic cells display progression of replication on damaged templates, allowing replication to be completed and averting replication fork collapse (12). Direct bypass of the fork-blocking lesion, permitting replication to proceed with little delay, would be an attractive mechanism to release an arrested replicon. Several early studies using bacteria and mammalian cells, however, have indicated that replicating cells exposed to UV light initially synthesize smaller DNA strands than undamaged cells (reviewed in reference 32). At a later stage, these molecules are converted into DNA of high molecular weight, possibly via late, postreplicative, filling of the lesion-containing gap by socalled postreplication repair. The presence of single-stranded gaps in both sister chromatids behind a replication fork was recently visualized by electron microscopy on DNA of UVexposed budding yeast Saccharomyces cerevisiae (34).Damage avoidance and translesion synthesis are two pathways that allow cells to replicate damaged templates (3, 12). Damage avoidance (also called template switching-dependent synthesis) uses the undamaged sister chromatid as a template. This can be achieved by replication fork regression or by strand invasion with the sister chromatid and results in error-free bypass of DNA lesions (7, 65). Translesion synthesis, on the other hand, is characterized by insertion of a nucleotide opposite the lesion by specialized DNA polymerases of the Y family (48). The reduced stringency of the active site and a lack of proofreading activity of translesion synthesis polymerases imply that translesion synthesis is an inherently mutagenic process.The Y family poly...

Multiple two-polymerase mechanisms in mammalian translesion DNA synthesis

“…Alternatively, the fork may be arrested at the lesion. [2][3][4][5][6] In each of these cases the error-free excision repair mechanisms, which rely on the complementary strand, cannot act to remove the lesion, since it is located on single-stranded DNA. To resolve this situation, and enable completion of DNA replication, DNA damage tolerance mechanisms bypass the lesion, without removing it from DNA, leading to restoration of the double-stranded structure of DNA.…”

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

“…In this sensitive model (i) UV dosimetry is not affected by melanin, and the absence of fur excludes shaving-induced artifacts (10); (ii) a very low, subtoxic, UV dose suffices to efficiently induce skin cancer with short latency (10, 11, reviewed in ref. 12); and (iii) the NER deficiency further increases the dependence on error-prone TLS of photolesions (7). The Rev1 B/B mutation only slightly increased the UV sensitivity of the Xpc Ϫ/Ϫ skin as the minimal erythema/edema (toxic) dose (MED; 13) was reduced from 500 J/m 2 to 350 J/m 2 .…”

“…Although the protein can incorporate deoxycytosines opposite abasic nucleotides and some damaged guanines, Rev1 plays a regulatory, rather than a catalytic, role in error-prone TLS of other damages, including photolesions (5-7). The role of Rev1 in a particularly error-prone subpathway of TLS of photolesions is mediated by its N-terminal BRCT domain (6,7). In agreement, mouse embryonic stem cells with a disruption of this domain (Rev1 B/B cells) lack all UV-induced nucleotide transversion mutations and part of the transitions (8).…”

“…In agreement, mouse embryonic stem cells with a disruption of this domain (Rev1 B/B cells) lack all UV-induced nucleotide transversion mutations and part of the transitions (8). Nevertheless, this Rev1 allele is hypomorphic as in Rev1 B/B cells photolesions ultimately are replicated, in contrast to completely Rev1-deficient cells (7). Rev1 B/B mice display no spontaneous phenotypes, unlike completely Rev1-deficient mice, supporting the hypomorphic nature of the Rev1 B allele, also at endogenous DNA damages (8,9).…”

“…4A) (7). Perturbation of replication in UV-exposed MEFs (5 J/m 2 ) was determined as described ( Fig.…”