REV1 and polymerase ζ facilitate homologous recombination repair

Abstract

REV1 and DNA Polymerase ζ (REV3 and REV7) play important roles in translesion DNA synthesis (TLS) in which DNA replication bypasses blocking lesions. REV1 and Polζ have also been implicated in promoting repair of DNA double-stranded breaks (DSBs). However, the mechanism by which these two TLS polymerases increase tolerance to DSBs is poorly understood. Here we demonstrate that full-length human REV1, REV3 and REV7 interact in vivo (as determined by co-immunoprecipitation studies) and together, promote homologous recombination repair. Cells lacking REV3 were hypersensitive to agents that cause DSBs including the PARP inhibitor, olaparib. REV1, REV3 or REV7-depleted cells displayed increased chromosomal aberrations, residual DSBs and sites of HR repair following exposure to ionizing radiation. Notably, cells depleted of DNA polymerase η (Polη) or the E3 ubiquitin ligase RAD18 were proficient in DSB repair following exposure to IR indicating that Polη-dependent lesion bypass or RAD18-dependent monoubiquitination of PCNA are not necessary to promote REV1 and Polζ-dependent DNA repair. Thus, the REV1/Polζ complex maintains genomic stability by directly participating in DSB repair in addition to the canonical TLS pathway.

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

“…Thus, survival is reduced when the expression of polζ is knocked down, and HDR does not seem to serve as an efficient backup under these conditions, as indicated by the lack of change in DNA sequence signature. A possible explanation is that polζ is involved also in HDR, as recently reported (32,33).…”

“…Although the identity of the DNA polymerase involved in the synthesis step of HDR is not clear, there were reports on the involvement of DNA polymerases η in an in vitro system (46) and of Rev1 and DNA polymerase ζ in cultured cells (32). The action of these or other polymerases in HDR is not expected to affect the sequence signatures that we have observed in our system, because they are expected to be distributed all over the synthesis patch, in contrast to the targeted and semitargeted base signatures that have been observed.…”

A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

“…This is based on our inability to detect altered DNA repair in cells depleted of RAD18 using siRNA approaches. 33 The RAD18 UBZ domain interferes with recruitment of multiple DDR factors to DNA repair foci Since the ability of excess RAD18 to interfere with 53BP1 recruitment to repair foci is dependent upon a functional UBZ domain, we tested whether expression of isolated UBDs from different DDR proteins can block chromatin spreading of other DDR factors. First, we verified that Flag-tagged RAD18 UBZ domain (191-238), RAP80 tandem UIMs (64-130), and the Pol η UBZ domain (615-670) bind to K63-linked ubiquitin in vitro (Fig.…”

“…Coverslips were stained for the indicated antibodies as described. 33,48 Briefly, the immunofluorescence staining protocol consisted of blocking for 30 min with 5% fetal bovine serum, 0.05% Triton X-100, and 1% goat serum, and then incubating coverslips with primary antibodies for 45 min. Coverslips were washed 3 times with PBS and then incubated with the appropriate secondary goat anti-rabbit or goat anti-mouse Alexa Fluor dye (488 or 595) conjugated secondary antibody (Molecular Probes) for 45 min, washed with PBS, counterstained with DAPI (Sigma) to visualize nuclear DNA, and then mounted onto slides with ProLong Gold antifade reagent (Invitrogen).…”

“…Cells were then transduced with AdNGUS24i adenovirus encoding I-SceI. Two days later, cells were subjected to flow cytometry analysis and mCherry-expressing cells were analyzed for I-SceI-induced GFP expression as described (Sharma et al, 2012). Recombination .…”