hRev7, putative subunit of hPolζ, plays a critical role in survival, induction of mutations, and progression through S-phase, of UV(254nm)-irradiated human fibroblasts

Abstract

Translesion synthesis (TLS) refers to mechanisms by which specialized DNA polymerases incorporate nucleotides opposite fork-blocking lesions and extend replication until standard replicative polymerases take over. The first eukaryotic TLS polymerase discovered, S. cerevisiae Polzeta, consists of catalytic subunit Rev3 and non-catalytic subunit Rev7. Human homologs of these two proteins have been identified. Studies by Lawrence, Maher, and colleagues comparing UV((254nm))-irradiated human fibroblast cell strains expressing high levels of hRev3 antisense to their normal parental strains demonstrated that there was no difference in cell survival, but that the frequency of UV-induced mutations in the derivative strains was 10-fold lower than that of the parental strains, indicating that hRev3 plays a critical role in such mutagenesis. To examine the role of hRev7 in TLS, we generated human fibroblasts expressing hRev7 siRNA, identified two derivative cell strains with significantly reduced levels of hRev7, and compared them to their parental strain and a vector control for cell survival, induction of mutations, and ability to traverse the cell cycle following exposure to UV radiation. Cells with reduced hRev7 were approximately 2-times more sensitive to UV-induced cytotoxicity than the controls, indicating that unlike hRev3, hRev7 plays a protective role for cells exposed to UV radiation. When these cell strains were assayed for the frequency of mutations induced by UV in their HPRT gene, cell stains with reduced hRev7 were 5-times less sensitive to UV-induced mutagenesis than control strains. In addition, when these four strains were synchronized at the G1/S border, released from the block, UV-irradiated, and allowed to traverse the cell cycle, the rate of progression through S-phase of the cell strains with reduced hRev7 was significantly slower than that of the control strains. These data strongly support the hypothesis that hRev7 is required for TLS past UV-photoproducts, and together with hRev3, comprise hPolzeta.

Rev7/Mad2B plays a critical role in the assembly of a functional mitotic spindle

“…21 The specificity of the Rev7 antibody used in this study was validated by various means as described previously. 28 The subcellular distribution of Rev7 was also confirmed in HCT116 cells and by using a previously-validated rabbit anti-Rev7 antibody 29 (data not shown).…”

“…Additionally, a previously-published siRNA sequence 5'-AGAA-GAAUCUUCAGUACUA-3' 46 was used to deplete RAN products (siRAN-4). For stable depletion, cells were transfected with a pSilencer vector either alone or expressing short hairpin against hRev7 (shRev7) 29 by using lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. The cells were selected with 1 mg/ml puromycin and selected clones were tested for the depletion efficiency using Western blotting.…”

The REV7 Subunit of DNA Polymerase ζ Is Essential for Primordial Germ Cell Maintenance in the Mouse

“…It has been reported that REV7 interacts with REV3 forming Pol , a DNA polymerase involved in translesion DNA synthesis, and suppression of REV7 impairs DNA damage tolerance in human fibroblasts (19,20,23). During embryogenesis, endogenous factors, such as metabolites, oxygen radicals, and nitrogen species, induce various types of DNA damage, and an effective DNA repair system is very important for normal development of organisms (46 -49).…”

“…REV7 also interacts with human REV1, but the biological significance of REV7 association with REV3 and REV1 in humans is unclear (20 -22). Human fibroblasts with siRNAmediated depletion of REV7 expression show high sensitivity to UV-induced cytotoxicity and reduced sensitivity to UV-induced mutagenesis compared with those in control lines, indicating that REV7 is required for tolerance to UV-induced DNA damage (23). Recently, it has been reported that Pol and REV1 are involved in homologous recombination repair of DNA double strand breaks, although its mechanism is not fully understood (24,25).…”

REV7 is required for anaphase-promoting complex-dependent ubiquitination and degradation of translesion DNA polymerase REV1

“…24 Similar findings were also obtained in REV7-compromised chicken DT40 cells and human foreskin fibroblasts, indicating a conservative role of REV7 in TLS in eukaryotes. 25,26 REV7 is also called MAD2B because it shares high sequence homology with MAD2, a key regulator in mitotic checkpoint. 27 Although REV7 does not bind MAD1, an anchoring protein for MAD2 at the kinetochore at the onset of checkpoint activation, 28 REV7, like MAD2, is capable of binding CDH1 and/or CDC20, which are coactivators of anaphase-promoting complex (APC), to impede APC activation.…”