Abstract
Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase epsilon. This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism. The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase epsilon that eliminates its polymerase and exonuclease activities. It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures. This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3'- to 5'-exonuclease-deficient mutant of DNA polymerase delta in a haploid cell. These results suggest that the catalytic activity of DNA polymerase epsilon participates in the same pathway as DNA polymerase delta, and this is consistent with the observation that DNA polymerases delta and epsilon colocalize in some punctate foci on yeast chromatids during S phase. The pol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast.
Highlights
Saccharomyces cerevisiae has three DNA polymerases1 that are required for cell growth, chromosomal DNA replication [1], and DNA double-strand break repair [2]. pol␣ consists of four subunits (Pol1p (Cdc17p), Pol10p, Pri1p, and Pri2p) and is primarily involved in the initiation of DNA replication and priming of Okazaki fragments. pol␦ and -⑀ are required during synthesis of the leading and lagging strands at the replication fork, binding at/or near replication origins, and moving along DNA with the replication fork [3, 4]
The pol2-16 mutant senesces more rapidly than wild type strain and has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast
The results show that the catalytic domain of pol⑀ participates in the same pathway as pol␦ and is required for rapid, efficient, and accurate chromosomal DNA replication in S. cerevisiae
Summary
Yeast Strains—The yeast S. cerevisiae strains used in this study are as follows: W303-1A (MATa ade can100 his3-11, -15 leu 112 trp ura3) Cells were suspended in fresh YPD medium containing 30 ng of ␣-factor/ml and incubated for 3 h at 25 °C. ␣-Factor was removed by centrifugation, and cells were resuspended at 1.3 ϫ 107 cells/ml in YPD containing 0.1 g/ml actinase E (Kakenseiyaku, Japan) and incubated at 16 °C. ␣-Factor was removed by centrifugation, and the cells were resuspended at 1.3 ϫ 107 cells/ml in YEPR containing 0.1 g/ml actinase E and incubated at 25 °C. The soluble fraction of the whole-cell extract was discarded, and the insoluble pellet was resuspended in 0.5 ml of lysis buffer This chromatin-containing suspension was sonicated to yield an average DNA size of 500 bp, clarified by centrifugation, and subjected to immunoprecipitation with rabbit anti-S. cerevisiae PCNA conjugated with protein G-Sepharose beads (Amersham Biosciences). Other Materials and Methods—Other materials and methods used in this study were described previously [4, 25]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
