No 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...

The RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were cloned by the polymerase chain reaction. DNA sequence analysis revealed an open reading frame of 418 amino acids for the human RAD52 homolog and of 420 amino acid residues for the mouse counterpart. The identity between the two proteins is 69% and the overall similarity 80%. The homology of the mammalian proteins with their counterparts from yeast is primarily concentrated in the N-terminal region. Low amounts of RAD52 RNA were observed in adult mouse tissues. A relatively high level of gene expression was observed in testis and thymus, suggesting that the mammalian RAD52 protein, like its homolog from yeast, plays a role in recombination. The mouse RAD52 gene is located near the tip of chromosome 6 in region G3. The human equivalent maps to region p13.3 of chromosome 12. Until now, this human chromosome has not been implicated in any of the rodent mutants with a defect in the repair of double-strand breaks.

The imaginal disk cells of Drosophila have a cell cycle that is very similar to that of mammalian cells. Data concerning factors inducing tumors in these cells may directly relate to the risk of these factors for inducing cancer in humans. One of the genes involved in the regulation of cell cycle control is wts (warts), the Drosophila homolog of the mammalian tumor suppressor gene LATS1. The Drosophila wts mutations are recessive lethal. However, homozygous clones that arise in heterozygous flies in the imaginal disk cells lead to epithelial tumors, spectacular outgrowths visible on the cuticle of the adult. We have treated Drosophila larvae, heterozygous for wts, with the chemical mutagen MMS (methyl methanesulfonate) or with X-rays and measured the appearance of epithelial tumors in the eclosing adult flies. This test is a variation of the well-known Drosophila somatic mutation and recombination test (SMART), where mostly recessive markers have been used leading to visible phenotypes in the eyes and wings of the fly. We show that the sensitivity of this test is far greater than the comparable test system using the recessive eye marker white.

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRAD54-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution.Double-strand breaks (DSBs) in DNA are induced by ionizing radiation and by various chemical compounds, such as free radicals and methyl methanesulfonate (MMS). In addition, DSBs arise as intermediates during V(D)J rearrangement in differentiating lymphocytes, transposition events, and meiotic recombination in germ cells. Unrepaired DSBs often lead to cell death or contribute to the formation of chromosomal aberrations such as deletions and translocations. In eukaryotes, DSBs can be repaired via two main pathways: (i) recombinational repair, which is dependent on the presence of an intact duplicate DNA sequence, and (ii) end-to-end rejoining, which is based on rejoining of the two DNA ends (46). The available evidence suggests that both mechanisms are conserved in eukaryotes from yeasts to humans. However, the relative contributions of both mechanisms in repair of DSBs differ considerably between lower and higher eukaryotes. Repair of DSBs in mammals has been investigated with radiation-sensitive rodent cell lines. Several of these cell lines have defects in repairing radiation-induced DSBs and are also strongly impaired in V(D)J rearrangement. Further studies have revealed that these mutants have defects in the end-to-end rejoining pathway (reviewed in references 26, 47, and 63). Repair of DSBs at defined sites in the genome of mammalian cells also occurs more frequently by end-to-end rejoining than by homologous recombination (21, 33). The same observations have been made in experiments studying the fate of linear plasmid molecules in mammalian cells and Xenopus laevis oocytes (27,29,31). Therefore, DSBs in higher eukaryotes appear to be repaired primarily by end-to-end rejoining mechanisms.In lower eukaryotes, however, repair of DSBs occurs almost exclusively by...