Christopher W. Lawrence

The Y-Family of DNA Polymerases

We would like to thank Tomoo Ogi for generating the unrooted phylogenetic tree shown in Figure 1Figure 1 and Junetsu Ito for his comments on our proposal.

Thymine-Thymine Dimer Bypass by Yeast DNA Polymerase ζ

The REV3 and REV7 genes of the yeast Saccharomyces cerevisiae are required for DNA damage-induced mutagenesis. The Rev3 and Rev7 proteins were shown to form a complex with DNA polymerase activity. This polymerase replicated past a thymine-thymine cis-syn cyclobutane dimer, a lesion that normally severely inhibits replication, with an efficiency of ∼10 percent. In contrast, bypass replication efficiency with yeast DNA polymerase α was no more than 1 percent. The Rev3-Rev7 complex is the sixth eukaryotic DNA polymerase to be described, and is therefore called DNA polymerase ζ.

Evidence for a second function for Saccharomyces cerevisiae Rev1p.

The function of the Saccharomyces cerevisiae REV1 gene is required for translesion replication and mutagenesis induced by a wide variety of DNA‐damaging agents. We showed previously that Rev1p possesses a deoxycytidyl transferase activity, which incorporates dCMP opposite abasic sites in the DNA template, and that dCMP insertion is the major event during bypass of an abasic site in vivo. However, we now find that Rev1p function is needed for the bypass of a T–T (6–4) UV photoproduct, a process in which dCMP incorporation occurs only very rarely, indicating that Rev1p possesses a second function. In addition, we find that Rev1p function is, as expected, required for bypass of an abasic site. However, replication past this lesion was also much reduced in the G‐193R rev1‐1 mutant, which we find retains substantial levels of deoxycytidyl transferase activity. This mutant is, therefore, presumably deficient principally in the second, at present poorly defined, function. The bypass of an abasic site and T–T (6–4) lesion also depended on REV3 function, but neither it nor REV1 was required for replication past the T–T dimer; bypass of this lesion presumably depends on another enzyme.

Cellular roles of DNA polymerase ζ and Rev1 protein

Abstract The majority of both spontaneous and DNA damage-induced mutations in eukaryotes result from replication processes in which DNA polymerase ζ (Polζ) and Rev1 protein (Rev1p) play major roles. Understanding these roles is likely to provide information relevant to the origin of genetic diseases, such as cancer, and may provide new insights for their prevention. DNA Polζ also appears to be involved in the somatic hypermutability that occurs during development of the immune response. The results from a variety of genetic and enzymological investigations have started to delineate the cellular roles of these enzymes, but a number of important issues have not yet been resolved and much remains to be learned. Questions concerning the possible existence of other subunits to these enzymes, of their possible association with one another or with other proteins, of the nature of their enzymatic activities and of the relative roles played by these and other DNA polymerases in the bypass of different kinds of DNA damage, require further investigation. Finally, very little is known about the way these enzymes are regulated and brought into play when needed.

Specificity and frequency of ultraviolet-induced reversion of an iso-1-cytochrome c ochre mutant in radiation-sensitive strains of yeast☆

The basis for the specific pattern of ultraviolet-induced reversion of cyc1-9, an ochre allele of the structural gene for iso-1-cytochrome c, has been examined in radiation-sensitive strains of yeast. Previous analysis, using RAD+ strains, showed that 21 out of 23 cyc1-9 revertants induced by ultraviolet light arose by A · T to G · C transition at the first position in the UAA codon, the remaining two occurring by A · T to T · A transversion at the second position (Stewart et al., 1972; Sherman & Stewart, 1974). All possible base-pair substitutions could be obtained with the aid of other mutagens. It has now been shown that this specificity depends largely on the action of the RAD6 locus, since ultraviolet-induced revertants of cyc1-9 arose by a variety of base-pair substitutions in a strain carrying the rad6-1 allele. Induced reversion frequencies in strains carrying this allele are much lower than normal, though significantly higher than the spontaneous frequency, and the strains are more sensitive to the lethal effects of both ultraviolet and X-irradiation. The phenotypically similar rad18-2 mutation, which appears to block the same repair pathway as rad6-1, also has some effect on the reversion specificity, but its action depends on the presence of other, unidentified, mutations. Specificity was, however, completely unaltered in an excision-defective strain carrying the rad1-2 allele. Induced reversion frequency of cyc1-9 was much higher than normal in this strain. Photoreactivation studies indicated that pyrimidine dimers were responsible for most of the revertants in RAD+, rad1 and rad6 strains. These experiments show that the RAD6+ locus is intimately concerned with error-prone repair, and suggest that excision repair is substantially error-free.

Ultraviolet-induced reversion of cyc1 alleles in radiation-sensitive strains of yeast: I. rev1 mutant strains☆

Radiation-sensitive strains of the yeast, Saccharomyces cerevisiae, that carry the rev1-1 mutation exhibit greatly reduced frequencies of reversion of a number of auxotrophic mutations (Lemontt, 1971a), much lower frequencies of forward mutation to auxotrophy (Lemontt, 1972) and substantially reduced frequencies of reversion of cyc1-9 (Lawrence and Christensen, 1976), an ochre allele of the structural gene for iso-1-cytochrome c (Stewart et al., 1972), when exposed to ultraviolet light. In contrast, the u. v. -induced reversion of cyc1-131, whose message contains the valine codon GUG in place of the AUG initiation codon (Stewart et al., 1971), is unaffected by the presence of the rev1-1 mutations (Lawrence and Christensen, 1976). We have confirmed this observation using the rev1-1 as well as the rev1-1 mutation and have also identified four other cyc1 alleles out of 15 examined that behave in the same way. It is clear, therefore, that while the REV1 gene function is required for the production of many mutational alterations at this locus, it is not for the production of certain specific events. The reason for this appears to depend on the genetic nature of these events rather than the kind of premutational lesion but, despite almost complete information on the base-pair changes and surrounding nucleotide sequences at these sites, it has not yet been possible to define their special nature precisely, indicating that the mutational process is surprisingly complex. The REV1 gene function is not required for the production of most base-pair additions or deletions and although necessary for the formation of most base-pair substitutions it is also not required for the substitutions that lead to the reversion of cyc1-131 and the proline missense mutant, cyc1-115. The REV1 gene function is necessary to produce identical substitutions at other sites, however, and also to produce different substitutions at the cyc1-131 site, suggesting that the special character of cyc1-115 and cyc1-131 is the requirement for a specific base-pair alteration at a specific site. Whatever the explanation, the evidence of this kind of allele-specific control of u.v. -induced reversion must be accounted for by any proposed model of the mutagenic process and must also be accommodated by any scheme to test environmental mutagens.

Cellular Functions of DNA Polymerase ζ and Rev1 Protein

Publisher Summary This chapter focuses on DNA polymerase ζ (Pol ζ) and Rev1 protein (Rev1p), which perform a variety of important and essential functions in eukaryotes, including roles in DNA damage tolerance, in the development of diversity among immunoglobulin genes, and in the repair of double-strand breaks by homologous recombination. The chapter reviews information about the properties and functions of Pol ζ and Rev1p. Most of these data come from yeast, but important results have also been obtained from a variety of other species, including the mouse, humans, and chickens. A combination of investigations over the past several years, examining the enzymology, genetics, and cell biology of Pol ζ and Rev1p, has contributed in the understanding of cellular functions. Different models proposed for Pol ζ and Rev1p functions are also examined in the chapter.

Specific Binding of Human MSH2·MSH6 Mismatch-Repair Protein Heterodimers to DNA Incorporating Thymine- or Uracil-containing UV Light Photoproducts Opposite Mismatched Bases

Previous studies have demonstrated recognition of DNA-containing UV light photoproducts by bacterial (Feng, W.-Y., Lee, E., and Hays, J. B. (1991) Genetics 129, 1007–1020) and human (Mu, D., Tursun, M., Duckett, D. R., Drummond, J. T., Modrich, P., and Sancar, A. (1997) Mol. Cell. Biol. 17, 760–769) long-patch mismatch-repair systems. Mismatch repair directed specifically against incorrect bases inserted during semi-conservative DNA replication might efficiently antagonize UV mutagenesis. To test this hypothesis, DNA 51-mers containing site-specific T-Tcis-syn-cyclobutane pyrimidine-dimers or T-T pyrimidine-(6-4′)pyrimidinone photoproducts, with all four possible bases opposite the respective 3′-thymines in the photoproducts, were analyzed for the ability to compete with radiolabeled (T/G)-mismatched DNA for binding by highly purified human MSH2·MSH6 heterodimer protein (hMutSα). Both (cyclobutane-dimer)/AG and ((6-4)photoproduct)/AG mismatches competed about as well as non-photoproduct T/T mismatches. The two respective pairs of photoproduct/(A(T or C)) mismatches also showed higher hMutSα affinity than photoproduct/AA “matches”; the apparent affinity of hMutSα for the ((6-4)photoproduct)/AA-“matched” substrate was actually less than that for TT/AA homoduplexes. Surprisingly, although hMutSα affinities for both non-photoproduct UU/GG double mismatches and for (uracil-cyclobutane-dimer)/AG single mismatches were high, affinity for the (uracil-cyclobutane-dimer)/GG mismatch was quite low. Equilibrium binding of hMutSα to DNA containing (photoproduct/base) mismatches and to (T/G)-mismatched DNA was reduced similarly by ATP (in the absence of magnesium).

REV7, a new gene concerned with UV mutagenesis in yeast

SummaryThree allelic mutations of a new yeast gene, which we have named REV7, have been isolated by testing 313 methyl methane sulfonate sensitive mutants for UV-induced reversion of a lys2 allele. Rev7 mutants are markedly deficient with respect to UV-induced reversion of lys2, are slightly sensitive to UV and appear to be in the RAD6 epistasis group for UV survival. Rev7-1, which is probably an amber mutation, does not appear to affect sporulation in homozygous diploids. The REV7 gene is located about 12 cM distal to HIS5 on chromosome IX.

hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody detection, it was not possible to demonstrate that the decrease in mutagenesis reflected decreased hRev3 protein. However, the conclusion is supported by the fact that in a similar study with a strain expressing a high level of antisense hREV1, a very similar result was obtained, i.e. UV or BPDE mutagenesis was virtually eliminated.