DNA lesions can often block DNA replication, so cells possess specialized low-fidelity, and often error-prone, DNA polymerases that can bypass such lesions and promote replication of damaged DNA. The Saccharomyces cerevisiae RAD30 and human hRAD30A encode Pol eta, which bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Here we show that a related human gene, hRAD30B, encodes the DNA polymerase Pol iota, which misincorporates deoxynucleotides at a high rate. To bypass damage, Pol iota specifically incorporates deoxynucleotides opposite highly distorting or non-instructional DNA lesions. This action is combined with that of DNA polymerase Pol zeta, which is essential for damage-induced mutagenesis, to complete the lesion bypass. Pol zeta is very inefficient in inserting deoxynucleotides opposite DNA lesions, but readily extends from such deoxynucleotides once they have been inserted. Thus, in a new model for mutagenic bypass of DNA lesions in eukaryotes, the two DNA polymerases act sequentially: Pol iota incorporates deoxynucleotides opposite DNA lesions, and Pol zeta functions as a mispair extender.

Abasic (AP) sites are one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA machinery. Here we show efficient bypass of an AP site by the combined action of yeast DNA polymerases ␦ and . In this reaction, Pol␦ inserts an A nucleotide opposite the AP site, and Pol subsequently extends from the inserted nucleotide. Consistent with these observations, sequence analyses of mutations in the yeast CAN1 s gene indicate that A is the nucleotide inserted most often opposite AP sites. The nucleotides C, G, and T are also incorporated, but much less frequently. Enzymes such as Rev1 and Pol may contribute to the insertion of these other nucleotides; the predominant role of Rev1 in AP bypass, however, is likely to be structural. Steady-state kinetic analyses show that Pol is highly inefficient in incorporating nucleotides opposite the AP site, but it efficiently extends from nucleotides, particularly an A, inserted opposite this lesion. Thus, in eukaryotes, bypass of an AP site requires the sequential action of two DNA polymerases, wherein the extension step depends solely upon Pol, but the insertion step can be quite varied, involving not only the predominant action of the replicative DNA polymerase, Pol␦, but also the less prominent role of various translesion synthesis polymerases. Abasic (AP) sites represent one of the most frequently formed DNA lesions in eukaryotes, and it has been estimated that a human cell loses as many as 10 4 purines per day from its genome (Lindahl and Nyberg 1972). In Saccharomyces cerevisiae, AP sites are efficiently repaired by the AP endonucleases encoded by the APN1 and APN2 genes. APN1 and APN2 provide alternate pathways for the removal of AP sites, and consequently, simultaneous inactivation of both the genes results in a dramatic decline in the efficiency to repair AP sites (Johnson et al. 1998).If the AP sites are not removed by excision repair processes, they present a block to the replication machinery. During replication, AP sites can be bypassed either by a specialized mutagenic DNA polymerase, or by errorfree mechanisms such as recombination or a copy-choice type of DNA synthesis. In S. cerevisiae, genes in the RAD6 epistasis group promote replication through DNA lesions (Prakash 1981). The REV1, REV3, and REV7 genes of this group are essential for UV-induced mutagenesis (Lawrence and Hinkle 1996), and these genes are also indispensable for mutagenesis induced by AP sites (Johnson et al. 1998). REV1 encodes a deoxycytidyl transferase activity (Nelson et al. 1996a), and the REV3-and REV7-encoded proteins together form DNA polymerase (Nelson et al. 1996b). Although Pol is absolutely required for damage-induced mutagenesis, and therefore for the mutagenic bypass of a variety of DNA lesions, our recent studies have indicated that on its own, Pol bypasses UV lesions very inefficiently (Johnson et al. 2000a). This is because Pol is very inefficient in inserting nucleotides opposite the 3Ј T of a cis-syn thymine-thym...

Oxidative damage to DNA has been proposed to have a role in cancer and ageing. Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA, and 7, 8-dihydro-8-oxoguanine (8-oxoG) is one of the adducts formed. Eukaryotic replicative DNA polymerases replicate DNA containing 8-oxoG by inserting an adenine opposite the lesion; consequently, 8-oxoG is highly mutagenic and causes G:C to T:A transversions. Genetic studies in yeast have indicated a role for mismatch repair in minimizing the incidence of these mutations. In Saccharomyces cerevisiae, deletion of OGG1, encoding a DNA glycosylase that functions in the removal of 8-oxoG when paired with C, causes an increase in the rate of G:C to T:A transversions. The ogg1Delta msh2Delta double mutant displays a higher rate of CAN1S to can1r forward mutations than the ogg1Delta or msh2Delta single mutants, and this enhanced mutagenesis is primarily due to G:C to T:A transversions. The gene RAD30 of S. cerevisiae encodes a DNA polymerase, Poleta, that efficiently replicates DNA containing a cis-syn thymine-thymine (T-T) dimer by inserting two adenines across from the dimer. In humans, mutations in the yeast RAD30 counterpart, POLH, cause the variant form of xeroderma pigmentosum (XP-V), and XP-V individuals suffer from a high incidence of sunlight-induced skin cancers. Here we show that yeast and human POLeta replicate DNA containing 8-oxoG efficiently and accurately by inserting a cytosine across from the lesion and by proficiently extending from this base pair. Consistent with these biochemical studies, a synergistic increase in the rate of spontaneous mutations occurs in the absence of POLeta in the yeast ogg1Delta mutant. Our results suggest an additional role for Poleta in the prevention of internal cancers in humans that would otherwise result from the mutagenic replication of 8-oxoG in DNA.

Summary Completion of DNA replication after replication stress depends on PCNA, which undergoes mono-ubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is poly-ubiquitinated to promote recombination dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by poly-ubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.