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...

Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.Progression of the replication fork is often impeded by DNA lesions caused by exogenous or endogenous DNA-damaging agents. Replication forks also stall when they encounter tightly bound proteins or aberrant DNA structures (7). Stalled replication forks activate the DNA damage tolerance pathway, which promotes the reinitiation of DNA synthesis with or without removing the replication-blocking lesion.Genetic studies in Saccharomyces cerevisiae indicate that Rad6 and Rad18 play central roles in DNA damage tolerance pathway. This pathway is mediated by the protein products of RAD6, RAD18, RAD5, MMS2, and UBC13, as well as several other gene products (4, 9, 22). Rad6 is a ubiquitin E2-conjugating enzyme that forms a stable complex with Rad18, an E3 ligase that binds DNA (2, 3). The Ubc13-Mms2 heterodimer is also an E2-conjugating enzyme (14). Rad5 is a DNA-dependent ATPase that functions as an E3 ligase and associates with the Ubc13-Mms2 complex, recruiting this complex to chromatin in response to DNA damage (27). Previous study demonstrated that PCNA is a substrate for RAD6-dependent ubiquitination (13). When DNA synthesis on one or both strands is arrested by DNA damage, PCNA is mono-ubiquitinated on lysine 164 (Lys164) by the Rad6-Rad18 complex. Mono-ubiquitinated PCNA may target stalled replication forks to initiate error-prone DNA repair via translesion DNA synthesis, a process that requires low-fidelity polymerases, Pol or Pol, to synthesize across the damage (10,15,25,29). Alternatively, the Ubc13-Mms2 complex and Rad5 modulate the polyubiquitination of Lys164 through lysine 63-linked ubiquitin chains. When modified in this manner, PCNA promotes error-free DNA repair (13,25).PCNA is also sumoylated at Lys164 (and secondarily at Lys127) via a mechanism dependent on a distinct E2-conjugating enzyme, Ubc9, and an E3 ligase, Siz1 (13). Although ubiquitination and sumoylation both occur on Lys164, there is evidence suggesting that the two modifications do not appear to have antagon...

DNA polymerase epsilon (Polepsilon) of Saccharomyces cerevisiae is purified as a complex of four polypeptides with molecular masses of >250, 80, 34 (and 31) and 29 kDa as determined by SDS-PAGE. The genes POL2, DPB2 and DPB3, encoding the catalytic Pol2p, the second (Dpb2p) and the third largest subunits (Dpb3p) of the complex, respectively, were previously cloned and characterised. This paper reports the partial amino acid sequence of the fourth subunit (Dpb4p) of Polepsilon. This protein sequence matches parts of the predicted amino acid sequence from the YDR121w open reading frame on S.cerevisiae chromosome IV. Thus, YDR121w was renamed DPB4. A deletion mutant of DPB4 (Deltadpb4) is not lethal, but chromosomal DNA replication is slightly disturbed in this mutant. A double mutant haploid strain carrying the Deltadpb4 deletion and either pol2-11 or dpb11-1 is lethal at all temperatures tested. Furthermore, the restrictive temperature of double mutants carrying Deltadpb4 and dpb2-1, rad53-1 or rad53-21 is lower than in the corresponding single mutants. These results strongly suggest that Dpb4p plays an important role in maintaining the complex structure of Polepsilon in S.cerevisiae, even if it is not essential for cell growth. Structural homologues of DPB4 are present in other eukaryotic genomes, suggesting that the complex structure of S. cerevisiae Polepsilon is conserved in eukaryotes.

Early in eukaryotic cell cycle, a pre‐RC is assembled at each replication origin with ORC, Cdc6, Cdt1 and Mcm2‐7 proteins to license the origin for use in the subsequent S phase. Licensed origin must then be activated by S‐Cdk and Ddk. At the onset of S phase, RPA is loaded on to the ARS in a reaction stimulated by S‐Cdk and Ddk, followed by Cdc45‐dependent loading of pol α, ‐δ, and ‐ɛ. This study examines cell cycle‐dependent localization of pol α, ‐δ and ‐ɛ in Saccharomyces cerevisiae using immuno‐histochemical and chromatin immuno‐precipitation methods. The results show that pol α, ‐δ, or ‐ɛ localizes on chromatin as punctate foci at all stages of the cell cycle. However, some foci overlap with or are adjacent to foci pulse‐labeled with bromodeoxyuridine during S phase, indicating these are replicating foci. DNA microarray analysis localized pol α, ‐δ, and ‐ɛ to early firing ARSs on yeast chromosome III and VI at the beginning of S phase. These data collectively suggest that bidirectional replication occurs at specific foci in yeast chromosomes and that pol α, ‐δ, and ‐ɛ localize and function together at multiple replication forks during S phase.

The ESC2 gene encodes a protein with two tandem C-terminal SUMO-like domains and is conserved from yeasts to humans. Previous studies have implicated Esc2 in gene silencing. Here, we explore the functional significance of SUMO-like domains and describe a novel role for Esc2 in promoting genome integrity during DNA replication. This study shows that esc2D cells are modestly sensitive to hydroxyurea (HU) and defective in sister chromatid cohesion and have a reduced life span, and these effects are enhanced by deletion of the RRM3 gene that is a Pif1-like DNA helicase. esc2D rrm3D cells also have a severe growth defect and accumulate DNA damage in late S/G 2 . In contrast, esc2D does not enhance the HU sensitivity or sister chromatid cohesion defect in mrc1D cells, but rather partially suppresses both phenotypes. We also show that deletion of both Esc2 SUMO-like domains destabilizes Esc2 protein and functionally inactivates Esc2, but this phenotype is suppressed by an Esc2 variant with an authentic SUMO domain. These results suggest that Esc2 is functionally equivalent to a stable SUMO fusion protein and plays important roles in facilitating DNA replication fork progression and sister chromatid cohesion that would otherwise impede the replication fork in rrm3D cells.