SummaryUpon blockage of chromosomal replication by DNA lesions, Y-family polymerases interact with monoubiquitylated proliferating cell nuclear antigen (PCNA) to catalyse translesion synthesis (TLS) and restore replication fork progression. Here, we assessed the roles of Arabidopsis thaliana POLH, which encodes a homologue of Y-family polymerase g (Polg), PCNA1 and PCNA2 in TLS-mediated UV resistance. A T-DNA insertion in POLH sensitized the growth of roots and whole plants to UV radiation, indicating that AtPolg contributes to UV resistance. POLH alone did not complement the UV sensitivity conferred by deletion of yeast RAD30, which encodes Polg, although AtPolg exhibited cyclobutane dimer bypass activity in vitro, and interacted with yeast PCNA, as well as with Arabidopsis PCNA1 and PCNA2. Co-expression of POLH and PCNA2, but not PCNA1, restored normal UV resistance and mutation kinetics in the rad30 mutant. A single residue difference at site 201, which lies adjacent to the residue (lysine 164) ubiquitylated in PCNA, appeared responsible for the inability of PCNA1 to function with AtPolg in UV-treated yeast. PCNA-interacting protein boxes and an ubiquitin-binding motif in AtPolg were found to be required for the restoration of UV resistance in the rad30 mutant by POLH and PCNA2. These observations indicate that AtPolg can catalyse TLS past UV-induced DNA damage, and links the biological activity of AtPolg in UV-irradiated cells to PCNA2 and PCNA-and ubiquitinbinding motifs in AtPolg.
The loss of bases from cellular DNA occurs via both spontaneous and mutagen-induced reactions. The resulting apurinic/apyrimidinic (AP) sites are cytotoxic and mutagenic but are counteracted by repair initiated by AP endonucleases. Previously, in vitro and bacterial transfection studies suggested that AP sites often prompt insertion of dAMP residues during replication, the A-rule. Dissimilar results have been obtained by transfecting DNA into eukaryotic cells. It seemed possible that these differences might be due to idiosyncrasies of transfection or aberrant replication of the transfecting DNA. The observation that AP endonuclease-deficient strains of the yeast Saccharomyces cerevisiae have elevated spontaneous mutation rates allowed us to determine the mutational specificity of endogenously generated AP sites in nuclear DNA. With the yeast SUP4-o gene as a mutational target, we found that a deficiency in the major yeast AP endonuclease, Apnl, provoked mainly single base-pair substitution; the rate of transposon Ty insertion was also enhanced. The rate of transversion to a G-C pair was increased 10-fold in Apnl-deficient yeast, including a 59-fold increase in the rate of AFT -+ CG events. In contrast, the rate of transversion to an AFT pair was increased by only 3-fold. A deficiency in N3-methyladenine glycosylase offset these substitution rate increases, indicating that they are due primarily to AP sites resulting from glycosylase action. Thus, the A-rule does not seem to apply to the mutagenic processing of endogenous abasic sites in S. cereviswae. Other results presented here show that AP endonuclease-deficient Escherichia coi exhibit a mutator phenotype consistent with the A-rule.The loss of DNA bases to form apurinic/apyrimidinic (AP) sites occurs constantly. Such abasic lesions result from the inherent chemical instability of DNA, as well as the incessant assault of endogenous and environmental agents (1). AP sites are formed directly through the slow hydrolysis of the N-glycosyl bonds that anchor bases to DNA, but pyrimidines are lost at a much slower rate than purines (2). Spontaneous and mutagen-induced DNA damages include modified bases that have unstable glycosyl bonds or are substrates for DNA glycosylases, enzymes that remove the damaged bases to generate AP sites (3, 4). AP sites in DNA provide blocks to DNA synthesis and are substrates for mutagenesis in both prokaryotes and eukaryotes (3).The onslaught of AP sites is opposed primarily by the class of repair activities called AP endonucleases (5-8). The predominant forms of these enzymes hydrolyze the phosphodiester bond on the immediate 5' side of an AP site in duplex DNA; the nicked molecule is then further processed by nucleases that remove the dangling 5' AP site, followed by DNA repair synthesis and ligation (4). AP endonucleases and the genes that encode them have been isolated from bacteria, the yeast Saccharomyces cerevisiae, Drosophila melanogaster, and mammalian cells (5-11). In Escherichia coli, exonuclease III and endonuclease...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.