Involvement of Mouse Rev3 in Tolerance of Endogenous and Exogenous DNA Damage

Abstract

The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase that is implicated in mutagenic translesion synthesis of damaged DNA. To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3. Although some strain-dependent variation was observed, Rev3 ؊/؊ embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities. Rev3 ؊/؊ cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability. Histochemical analysis of Rev3 ؊/؊ embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages. Although increased levels of p53 are detected in Rev3 ؊/؊ embryos, the embryonic phenotype was not rescued by the absence of p53. A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3 ؊/؊ embryos. The inner cell mass of cultured Rev3 ؊/؊ blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene. These combined data are compatible with a model in which, in the absence of polymerase , double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response. Together, these data are consistent with involvement of polymerase in translesion synthesis of endogenously and exogenously induced DNA lesions.In cancer genetics, the paradigm holds the sequential mutation of a series of oncogenes and tumor suppressor genes responsible for the evolutionary development of a normal cell into a fully malignant, metastasizing tumor (67). Most mutations are induced by nucleotide damage, originating from endogenous sources or inflicted by exogenous agents (49). Nucleotide damage that is not removed by DNA repair proteins generally leads to an arrest of the replication fork, due to the rigidity of the replicative polymerases, preventing incorporation of a nucleotide opposite a damaged template (51). To escape this arrest, cells possess multiple pathways that enable the completion of DNA replication despite the presence of replication-blocking DNA damage (3, 9).Considerable progress has recently been made in identifying the actors in translesion synthesis, a pathway implicated in replicating damaged DNA. In both prokaryotes and eukaryotes, multiple polymerases have been identified that are capable of replicating DNA templates containing a variety of lesions. This enables the completion of replication and therefore safeguards cellular survival, albeit frequently at the expense of the introduction of mutations. Based on sequence homology and activity in vitro, most of the polymerases associated with translesion synthesis belong to the newly recognized Y superfamily of DNA polymerases (16,21,30,32,57,70). The heterodimeric Saccharomyces cerevisiae polymerase , comprised of the REV3 catalytic subun...

Loss of DNA Polymerase ζ Causes Chromosomal Instability in Mammalian Cells

“…Developmental and cellular proliferation defects due to loss of DNA repair genes have been ''rescued'' to different extents by simultaneously removing p53 (36)(37)(38). In contrast, we did not detect any effect of removal of p53 for development of Rev3L À/À embryos, a finding also reported by other groups (35,39 ;p53 À/À mice were isolated and cultured. Rev3L +/+ and Rev3L +/À fibroblasts proliferated immediately and similarly.…”

“…This suggests that pol~may be required to help cells tolerate endogenous DNA damage during mammalian development. Similarly, cells from Rev3L null embryos do not proliferate in vitro but remain quiescent with cell death occurring over time (26)(27)(28)35). The null Rev3L cells from midgestation embryos may have accumulated levels of unrepaired endogenous DNA damage that activate DNA damage checkpoints and preclude cell division in vitro.…”

“…A form of multicolor fluorescence in situ hybridization known as COBRA was used to examine metaphases from Rev3L À/À embryos, and the incidence of translocations seemed to be increased severalfold (35). The total numbers of rearrangements found were low, and different categories of translocations were not reported.…”

DNA polymerase zeta (pol ζ) in higher eukaryotes

“…Heterozygous Rev3L+/-embryos and mice are developmentally normal. The level of p53 protein was increased, and increased apoptosis was observed in embryos lacking Rev3L, probably as a response to genotoxic stress [79]. However, disruption of p53 is unable to rescue Rev3L null embryos [75,[79][80][81].…”

“…The uniform result, disrupting several different regions of the gene and in different types of ES cells and strain backgrounds, is that disruption of mouse Rev3L results in lethality during development. Rev3L null embryos do not survive past 8.5-12.5 days after fertilization and those that do survive to mid-gestation are delayed in their development [75][76][77][78][79]. Heterozygous Rev3L+/-embryos and mice are developmentally normal.…”