CloningDPB3, the gene encoding the third subunit of DNA polymerase II ofSaccharomyces cerevisiae

Abstract

DNA polymerase II purified from Saccharomyces cerevisiae contains polypeptides with apparent molecular masses of greater than 200, 80, 34, 30 and 29 kDa, the two largest of which (subunits A and B) are encoded by the essential genes POL2 and DPB2. By probing a lambda gt11 expression library of yeast DNA with antiserum against DNA polymerase II, we isolated a single gene, DPB3, that encodes both the 34- and 30-kDa polypeptides (subunit C and C'). The nucleotide sequence of DPB3 contained an open reading frame encoding a 23-kDa protein, significantly smaller than the observed molecular masses, 34- or 30-kDa, which might represent post-translationally modified forms of the DPB3 product. The predicted amino acid sequence contained a possible NTP-binding motif and a glutamate-rich region. NTP-binding motif and a glutamate-rich region. A dpb3 deletion mutant (dpb3 delta) was viable and yielded a DNA polymerase II lacking the 34- and 30-kDa polypeptides. dpb3 delta strains exhibited an increased spontaneous mutation rate, suggesting that the DPB3 product is required to maintain fidelity of chromosomal replication. Since a fifth, 29-kDa polypeptide was present in DNA polymerase II preparations from wild-type cell extracts throughout purification, the subunit composition appears to be A, B, C (or C and C') and D. The 5' nontranscribed region of DPB3 contained the MulI-related sequence ACGCGA, while the 0.9-kb DPB3 transcript accumulated periodically during the cell cycle and peaked at the G1/S boundary. The level of DPB3 transcript thus appears to be under the same cell cycle control as those of POL2, DPB2 and other DNA replication genes. DPB3 was mapped to chromosome II, 30 cM distal to his7.

Schizosacchromyces pombeDpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

“…Based on the observations that budding yeast DPB2 is required for chromosomal DNA replication (Araki et al, 1991a), we anticipated that S. pombe cells lacking dpb2 ϩ would also be defective for DNA synthesis. To test this possibility, we first analyzed DNA content in ⌬dpb2 germinating spores by FACS.…”

“…Deletion of each of the three smaller subunits from the S. cerevisiae genome results in different phenotypes. Only Dpb2 is essential for viability and normal S phase progression (Araki et al, 1991a). Although both Dpb3 and Dpb4 are not required for cell viability, deletion of the genes encoding these proteins does result in partial defects in DNA replication (Araki et al, 1991b;Ohya et al, 2000).…”

“…Only Dpb2 is essential for viability and normal S phase progression (Araki et al, 1991a). Although both Dpb3 and Dpb4 are not required for cell viability, deletion of the genes encoding these proteins does result in partial defects in DNA replication (Araki et al, 1991b;Ohya et al, 2000). Interestingly, both Dpb3 and Dpb4 contain histone-fold motifs, suggesting these proteins might be involved in chromatin remodeling (Araki et al, 1991b;Li et al, 2000;Ohya et al, 2000).…”

“…Although both Dpb3 and Dpb4 are not required for cell viability, deletion of the genes encoding these proteins does result in partial defects in DNA replication (Araki et al, 1991b;Ohya et al, 2000). Interestingly, both Dpb3 and Dpb4 contain histone-fold motifs, suggesting these proteins might be involved in chromatin remodeling (Araki et al, 1991b;Li et al, 2000;Ohya et al, 2000). Based on the existence of these motifs, it has been proposed these subunits might facilitate replication through heterochromatic regions of DNA (Fuss and Linn, 2002).…”

DNA polymerase ε encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe

“…This lack of complementation may be due to the complexity of Pole. Firstly, S. cerevisiae PolII consists of at least four polypeptide subunits , the 256-kDa Pol2 catalytic subunit (Morrison et al 1990), the second largest subunit (80 kDa) encoded by DPB2 (Araki et al 1991a), the third subunit (34 and 31 kDa) encoded by DPB3 (Araki et al 1991b), and the fourth subunit (29 kDa) encoded by DPB4 (our unpublished results), respectively. The subunit structure of S. pombe Pole is not known at the present time.…”