Structure and function of the fourth subunit (Dpb4p) of DNA polymerase epsilon in Saccharomyces cerevisiae

Abstract

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.

Figure 1

Partial purification of Polɛ complex from wild-type and Δdpb4 mutant yeast cells. Yeast cell extracts were prepared, applied to an S–Sepharose column and the proteins retained on the column were eluted with buffer A containing 0.5 M NaCl as described previously (11). The eluate was dialysed against buffer A, 0.1 M NaCl, applied to a Mono Q column equilibrated with the same buffer, and Polɛ was eluted with a linear gradient from 0.1 to 0.5 M NaCl in buffer A (11). Assays for the activity of Polɛ were carried out using oligo(dT)₁₀/poly(dA)_>500 (1:50) as a template-primer (A) and the fractions were analysed by SDS–PAGE and western blotting (6). Filters were probed with rabbit antibodies against Polɛ complex (21) (B) and Dpb4p (C). The purified Polɛ complex (11) (20 ng) was also used as a control (M) in (B) and (C). The numbers shown in the Figure are the fraction numbers after Mono Q column chromatography.

Figure 2

Δdpb4 mutant has a defect in S phase progression. Yeast strains [W303-1A (DPB4) and congenic W303-1A Δdpb4 strain (Δdpb4)] grown at 25°C in YPD medium to 1 × 10⁶ cells/ml were synchronised with α factor as described in Materials and Methods and separated into two equal portions. One was further incubated at 25°C and the other portion was incubated at 37°C. At various times, an aliquot was withdrawn, cells were collected by centrifugation, fixed with ethanol, stained with propidium iodide and analysed by FACScan as published (23). 1C and 2C, relative DNA content per cell; asy, before synchronisation.

Figure 3

Amino acid alignment of S.cerevisiae Dpb4p with homologues from other organisms. Amino acid sequence comparison between S.cerevisiae Dpb4p, S.pombe polypeptide (SpDpb4, accession no. CAB09118), C.elegans putative DNA binding protein (Ce, accession no. AAC77511), mouse CBF-A (Mouse, accession no. P25209), Xenopus nuclear transcription factor Y (Xenopus, accession no. AAC82336) and human nuclear transcription factor Y (Human, accession no. NP-006157) were carried out using the program ClustalW1.7 (32). Residues are shaded in red boxes to indicate sequence identity between either the S.cerevisiae Dpb4p or S.pombe Dpb4p homologue. Residues are shaded in light red to indicate sequence identity between other Dpb4 homologues. Dashes denote gaps in the sequence introduced to maximise the alignment. The conserved histone-fold motif is double overlined.