Mutator Phenotypes Caused by Substitution at a Conserved Motif A Residue in Eukaryotic DNA Polymerase δ

Ranga N Venkatesan,Jessica J Hsu,Nicole A Lawrence,Bradley D Preston,Lawrence A Loeb

doi:10.1074/jbc.m510245200

Ranga N Venkatesan, Jessica J Hsu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m510245200

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Abstract

Eukaryotic DNA polymerase (Pol) delta replicates chromosomal DNA and is also involved in DNA repair and genetic recombination. Motif A in Pol delta, containing the sequence DXXXLYPSI, includes a catalytically essential aspartic acid as well as other conserved residues of unknown function. Here, we used site-directed mutagenesis to create all 19 amino acid substitutions for the conserved Leu(612) in Motif A of Saccharomyces cerevisiae Pol delta. We show that substitutions at Leu(612) differentially affect viability, sensitivity to genotoxic agents, cell cycle progression, and replication fidelity. The eight viable mutants contained Ile, Val, Thr, Met, Phe, Lys, Asn, or Gly substitutions. Individual substitutions varied greatly in the nature and extent of attendant phenotypic deficiencies, exhibiting mutation rates that ranged from near wild type to a 37-fold increase. The L612M mutant exhibited a 7-fold elevation of mutation rate but essentially no detectable effects on other phenotypes monitored; the L612T mutant showed a nearly wild type mutation rate together with marked hypersensitivity to genotoxic agents; and the L612G and L612N strains exhibited relatively high mutation rates and severe deficits overall. We compare our results with those for homologous substitutions in prokaryotic and eukaryotic DNA polymerases and discuss the implications of our findings for the role of Leu(612) in replication fidelity.

Highlights

Pols ␦ and ⑀ synthesize DNA with high fidelity and are highly processive upon interaction with proliferating cell nuclear antigen (8 –10)
We exploited the evolutionary homology in the catalytic domains of family A and family B DNA polymerases to create mutants of Saccharomyces cerevisiae Pol ␦ that increase mutation rates
We demonstrate that amino acid substitutions at Leu612 in S. cerevisiae motif A differentially affect viability, mutation rates, cell cycle progression, and sensitivity to genotoxic agents

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains—Standard recombinant DNA and yeast molecular genetic techniques, including growth media, were followed [27, 28]. We replaced the entire chromosomal copy of POL3 in the strain BY4741 (MATa his leu2⌬ met15⌬ ura3⌬) (ATCC, Manassas, VA) with a KanMX cassette to yield BY4741-pol3⌬; POL3 function is provided by a wild type copy of the gene carried on the plasmid YCplac (ATCC), which contains URA3 selectable marker to obtain plasmid Ycplac33-POL3. Transformed cells were grown on SC-Leu-Ura plates [28] for 2–3 days to select for both the YCplac111-pol, (pol mutants, LEU2 marker) and pGL310 (POL3, wild type, URA3 marker) plasmids. The pGL310-POL3 plasmid was shuffled out by streaking 10 –20 colonies on SC-Leu [28] plates containing 5-fluoroorotic acid (ZymoResearch, Orange, CA; 1 mg/ml). The stained cells were diluted 10 times in 1ϫ phosphate-buffered saline, 1 mM EDTA, sonicated, and sorted on a BD Biosciences flow cytometer

RESULTS

DISCUSSION

Base substitutions