The DNA Polymerase Domain of polε Is Required for Rapid, Efficient, and Highly Accurate Chromosomal DNA Replication, Telomere Length Maintenance, and Normal Cell Senescence inSaccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase ⑀. 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 ⑀ 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 ␦ in a haploid cell. These results suggest that the catalytic activity of DNA polymerase ⑀ participates in the same pathway as DNA polymerase ␦, and this is consistent with the observation that DNA polymerases ␦ and ⑀ 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.Saccharomyces cerevisiae has three DNA polymerases (pol␣, -␦, and -⑀) 1 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 precise roles of pol␦ and pol⑀ during leading and lagging strand synthesis have yet not been defined; however, genetic and biochemical evidence suggests that lagging strand synthesis is carried out by pol␣ and pol␦ (5, 6). Nevertheless, simian virus 40 DNA replication only requires pol␣ and pol␦ (5).S. cerevisiae pol␦ contains the three subunits Pol3 (Cdc2), Hys2 (Pol31) (7), and Pol32 (8), which are homologues of Schizosaccharomyces pombe Pol3, Cdc1, and Cdc27, respectively. S. pombe pol␦ contains one additional subunit, Cmt1 (9). Purified yeast pol␦ requires accessory factors including PCNA and the RF-C to catalyze processive DNA synthesis; this suggests that pol␦ may be the leading strand DNA polymerase (5, 10). pol␦ has a 3Ј-to 5Ј-exonuclease, which acts as a proofreading/editing polymerase during DNA synthesis (11, 12).S. cerevisiae pol⑀ is also a multisubunit complex consisting of Pol2p, Dpb2p, Dpb3p, and Dpb4p (13,14). pol⑀ requires PCNA and RF-C complex to catalyze processive DNA synthesis on singly primed single-stranded viral DNA, although pol⑀ is a highly processive enzyme (13,15,16). Pol2p is the catalytic subunit of pol⑀, and it is encoded by the POL2 gene (17), which is essential in yeast. pol⑀ is a class B polymerase, characterized by six conserved domains (I-VI) in the N-terminal ha...

Structure and Function in the Budding Yeast Nucleus

“…Between 15 and 20 discrete foci were detected that were Origin Recognition Complex (ORC), S phase-, and origin-specific (Pasero et al 1997). The same type of foci were later observed by visualizing replisome components such as PCNA and DNA polymerase a or e by immunostaining or fluorescent tagging in living cells (Ohya et al 2002;Hiraga et al 2005;Kitamura et al 2006). Because the number of these foci is far smaller than the number of replication forks, they are thought to correspond to replication factories in which several elongating forks are grouped.…”

Mutagenic and Recombinagenic Responses to Defective DNA Polymerase δ Are Facilitated by the Rev1 Protein in pol3-t Mutants of Saccharomyces cerevisiae

“…The connection between dysfunctional DNA replication and genome instability has been firmly established in yeast by the elevated rates of many types of mutagenic and clastogenic events observed in a variety of DNA replication mutants (Aguilera and Klein 1988;Gordenin et al 1992;Ruskin and Fink 1993;Reagan et al 1995;Ohya et al 2002;Meyer and Bailis 2007). In particular, mutations that disrupt the polymerase function of Pol d confer increased rates of mutation and recombination (Gordenin et al 1992;Tran et al 1995Tran et al , 1996Tran et al , 1997Kokoska et al 1998Kokoska et al , 2000Lobachev et al 1998Lobachev et al , 2000Galli et al 2003), consistent with incom- 18.8 (9.3-24.1) 4.2 9.6 (6.7-11.4) 5.7 pol3-t rev7D…”

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

“…The POL2 gene of Pol e consists of two halves: the N-half encodes the active polymerase and the C-half comprises an inactive polymerase (30). The C-half of POL2 is essential to cell viability and binds the accessory factors Dpb2, -3, and -4, whereas the active N-terminal polymerase encoded by POL2 is not essential, although cells are severely compromised in S-phase progression (31)(32)(33). Our earlier chemical cross-linking MS study showed that the N-and C-halves of Pol2 each have numerous internal cross-links, yet there is only one cross-link between the two halves of Pol2, suggesting the N-and C-halves of Pol2 are distinct folding units connected by a linker region that separates them spatially (SI Appendix, Fig.…”