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.
Two forms of DNA polymerase II (⑀) of Saccharomyces cerevisiae, Pol II* and Pol II, were purified to near homogeneity from yeast cells. Pol II* is a four-subunit complex containing a 256-kDa catalytic polypeptide, whereas Pol II consists solely of a 145-kDa polypeptide derived from the N-terminal half of the 256-kDa polypeptide of Pol II*. We show that Pol II* and Pol II are indistinguishable with respect to the processivity and rate of DNA-chain elongation. The equilibrium dissociation constants of the complexes of Pol II* and Pol II with the DNA template showed that the stability of these complexes is almost the same. However, when the rates of dissociation of the Pol II* and Pol II from the DNA template were measured using single-stranded DNA as a trap for the dissociated polymerase, Pol II* dissociated 75-fold faster than Pol II. Furthermore, the rate of dissociation of Pol II* from the DNA template became faster as the concentration of the single-stranded DNA was increased. These results indicate that the rapid dissociation of Pol II* from the DNA template is actively promoted by single-stranded DNA. The dissociation of Pol II from the DNA template was also shown to be promoted by single-stranded DNA, although at a much slower rate. These results suggest that the site for sensing single-stranded DNA resides within the 145-kDa Nterminal portion of the catalytic subunit and that the efficiency for sensing single-stranded DNA by this site is positively modulated by either the C-terminal half of the catalytic subunit and/or the other subunits.In Saccharomyces cerevisiae, DNA polymerase II (⑀), as well as DNA polymerases I (␣) and III (␦), are required for DNA replication (1, 2). Deletion of the POL2 gene, which codes for the catalytic subunit of DNA polymerase II, causes cell death with a terminal dumbbell morphology, the hallmark of a defect in DNA replication (3). Direct measurements of in vivo DNA synthesis in temperature-sensitive pol2 mutants revealed that chromosomal DNA replication ceases at the restricted temperature (4, 5). The observation that a pol2 mutant defective in 3Ј35Ј exonuclease activity exhibits a mutator phenotype at a variety of genetic markers throughout the genome supports the idea that participation of the polymerase in chromosomal DNA replication is not restricted to certain sites on the genome (6). Recent work by Aparicio et al. (7) revealed that DNA polymerase II is recruited to the origin at the time of initiation of DNA replication and proceeds along the DNA with the replication fork. This finding, together with others, strongly supports the idea that DNA polymerase II is a component of the replication apparatus and that it is responsible for DNA synthesis on either the leading or lagging strand. However, whereas DNA polymerase I is responsible for laying down RNA-DNA primers, specific roles for DNA polymerases II and III at the fork have not been determined (8, 9).DNA repair is another cellular process for which the function of DNA polymerase II might be required. Among the DNA po...
Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C. We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C. The RFC2 gene is located on chromosome X of S. cerevisiae and is essential for cell growth. Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a defect in chromosomal DNA replication. The steady-state levels of RFC2 mRNA fluctuated less during the cell cycle than other genes involved in DNA replication. Nucleotide sequence of the gene revealed an open reading frame corresponding to a polypeptide with a calculated Mr of 39,716 and a high degree of amino acid sequence homology to the 37-kDa subunit of human RF-C. Polyclonal antibodies against bacterially expressed Rfc2 protein specifically reduced RF-C activity in the RF-C-dependent reaction catalyzed by yeast DNA polymerase III. Furthermore, the Rfc2 protein was copurified with RF-C activity throughout RF-C purification. These results strongly suggest that the RFC2 gene product is a component of yeast RF-C. The bacterially expressed Rfc2 protein preferentially bound to primed single-strand DNA and weakly to ATP.
We have previously shown that DNA polymerase ⑀ (Pol ⑀) of Saccharomyces cerevisiae binds stably to double-stranded DNA (dsDNA), a property not generally associated with DNA polymerases. Here, by reconstituting Pol ⑀ activity from Pol2p-Dpb2p and Dpb3p-Dpb4p, its two component subassemblies, we report that Dpb3p-Dpb4p, a heterodimer of histone-fold motif-containing subunits, is responsible for the dsDNA binding. Substitution of specific lysine residues in Dpb3p, highlighted by homology modeling of Dpb3p-Dpb4p based on the structure of the histone H2A-H2B dimer, indicated that they play roles in binding of dsDNA by Dpb3p-Dpb4p, in a manner similar to the histone-DNA interaction. The lysine-substituted dpb3 mutants also displayed reduced telomeric silencing, whose degree paralleled that of the dsDNA-binding activity of Pol ⑀ in the corresponding dpb3 mutants. Furthermore, additional amino acid substitutions to lysines in Dpb4p, to compensate for the loss of positive charges in the Dpb3p mutants, resulted in simultaneous restoration of dsDNA-binding activity by Pol ⑀ and telomeric silencing. We conclude that the dsDNA-binding property of Pol ⑀ is required for epigenetic silencing at telomeres.In eukaryotic cells, compaction of DNA into the higher order structure of chromatin involves many highly regulated steps and is required when cells go through S-phase in which the chromatin is temporarily unpacked for DNA replication (1). Epigenetic information on maintenance of both silenced and expressed states of chromatin must also be properly propagated during S-phase for these epigenetic states to be stably transmitted to subsequent generations (2). Thus, duplication of both chromosomal DNA and its chromatin states are tightly coupled processes. Proliferating cell nuclear antigen, one of the key components in the replication machinery serving as a platform for recruiting replication proteins (3), is considered to be a factor connecting DNA replication to chromatin assembly, most probably by directing chromatin assembly factor I to replicated DNA (4). As proliferating cell nuclear antigen mutants defective for chromatin assembly factor I interaction show reduced silencing (5), replication-coupled chromatin assembly mediated by these proteins is suggested to be a step required for proper inheritance of epigenetic chromatin structures.In Saccharomyces cerevisiae, DNA polymerase ⑀ (Pol ⑀), 4 another major component in DNA replication, has recently been shown to participate in the stable inheritance of the silenced state of chromatin (6). Pol ⑀ is a four-subunit complex comprising the catalytic subunit, Pol2p, and three auxiliary subunits, Dpb2p, Dpb3p, and Dpb4p (7-11); this subunit composition is conserved from yeast to humans (12-17). Among the auxiliary subunits, Dpb3p and Dpb4p contain a histone-fold motif (11, 16), a structural motif originally found in histones and involved in histone-histone, and histone-DNA interactions (18). The motifs in Dpb3p and Dpb4p are homologous in sequence to those in NF-YC and NF-YB (two sma...
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