Dpb11 controls the association between DNA polymerases alpha and epsilon and the autonomously replicating sequence region of budding yeast

Abstract

Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase epsilon (Dpb11-Polepsilon complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polepsilon associated with these fragments. Association of Dpb11 and Polepsilon with these fragments was mutually dependent, suggesting that the Dpb11-Polepsilon complex associates with the ARS. Moreover, Dpb11 was required for the association of Polalpha-primase with the fragments. Thus, it seems likely that association of the Dpb11-Polepsilon complex with the ARS fragments is required for the association of the Polalpha-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Polepsilon in dpb11-1 cells associated with early- and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases alpha and epsilon and the ARS.

FIG. 1

Dpb11 and Pol2 form a complex. (A) Cells of W303-1Ab, YHM011 (DPB11-9myc), and YHM01101 (POL2-3HA DPB11-9myc) were spheroplasted and treated with DSP (+) or left untreated (−). Lysates were prepared from log-phase cells (log) and cells arrested by α-factor (α), HU, and nocodazole (Noc) with the indicated genotypes: an epitope-tagged gene (+) and a wild-type allele (−). HA-tagged Pol2 was immunoprecipitated with anti-HA mouse MAb 12CA5. Extracts or immunoprecipitates were separated in a SDS–7.5% polyacrylamide gel and myc-tagged Dpb11 and HA-tagged Pol2 were detected by immunoblotting, using anti-myc rabbit polyclonal antibodies or anti-HA mouse MAb 16B12, respectively. (B) Extracts and immunoprecipitates from YHM011 (lanes 1 and 3) and YHM01101 (lanes 2 and 4) were separated in a SDS–7.5% polyacrylamide gel as described in panel A. In addition to Pol2 and Dpb11, Mcm2 and tubulin were detected by immunoblotting with anti-Mcm2 (yN-19; Santa Cruz Biotechnology) and anti-tubulin (YOL1/34) antibodies. (C) YHM01101, YHM31101 (mcm5-1), and YHA500 (dpb2-1 POL2-3HA DPB11-9myc) cells were grown at 25°C and then cultured at 36°C for 1 h. The cell lysates were subjected to immunoprecipitation using an anti-HA antibody.

FIG. 2

Neutral-neutral 2D gel analysis of the chromosomal ARS1 locus in synchronized cells. YHA410 (WT) and YHA411 (dpb11-1) cells were arrested in G₁ phase by α-factor and released to YPD medium at 37°C. The same number of cells at each time point (in minutes) was subjected to 2D gel electrophoresis and probed for the 5-kb NcoI fragment containing ARS1.

FIG. 3

Dpb11 associates with the ARS fragments in S phase. (A and B) YHM01 (POL2-3HA, WT) and YHM011 (DPB11-9myc, WT) cells were arrested in G₁ phase and released in YPD medium at 16°C. Cells were withdrawn from the culture every 15 min and fixed with formaldehyde. The chromatin fraction was sonicated and used for immunoprecipitation of HA- and myc-tagged proteins. PCR was performed either on immunoprecipitates derived from the same number of cells at each time point (in minutes) or on the 0-min chromatin fraction from the whole-cell extract (WCE). Note that faint signals of non-ARS fragments on the 90-min immunoprecipitates from YHM011 in panel B were not reproducibly obtained. (C) The DNA content was measured by FACS analysis of the samples collected in panel A. The percentage of budded cells is also shown.

FIG. 4

Association of the Dpb11 protein with the ARS fragments depends on the function of Mcm5, Rfa2, and Dpb2 but not on Cdc17 (Polα). YHM011 (DPB11-9myc) (A), YHM311 (DPB11-9myc mcm5-1) (B), YHM611 (DPB11-9myc rfa2-2) (C), YHA503 (DPB11-9myc dpb2-1) (D), YHM511 (DPB11-9myc cdc17-1) (E), and YHM501 (POL2-3HA cdc17-1) (F) cells were treated with α-factor for 3 h, 0.2 M HU was added, and the culture was incubated for another hour. The α-factor was removed, and the cells were suspended in YEPR medium containing 0.2 M HU at 36°C. PCR was performed as described in the legend to Fig. 3. In the presence of HU, the G₁ cells of all strains retained 1C DNA contents throughout the progression of budding (data not shown). Note that the signals appeared after release from α factor in panels B and D were not observed reproducibly.

FIG. 5

Association of Pol2 and Pri1 with ARS fragments requires Dpb11. (A to D). Association of Mcm4, Rfa1, Pol2, and Pri1 with ARS1- and ARS305-containing fragments in wild-type and dpb11-1 cells. YHM08 (MCM4-3HA) (A), YHM18 (MCM4-3HA dpb11-1) (A), YHM014 (RFA1-18myc) (B), YHM114 (RFA1-18myc dpb11-1) (B), YHM01 (POL2-3HA) (C), YHM11 (POL2-3HA dpb11-1) (C), YHM013 (PRI1-9myc) (D), and YHM113 (PRI1-9myc dpb11-1) (D) cells were arrested by α-factor and released to YEPR medium in the presence of HU at 36°C, as described in the legend to Fig. 4. PCR was performed as described in Fig. 3. (E) The DNA content of the cells were measured by FACS analysis after the cells had been cultured at 36°C without HU. In the presence of HU, the DNA content of cells remained exclusively 1C throughout the progression of budding.

FIG. 6

Pol2 associates with a late-firing origin-containing fragment as well as with a early-firing origin-containing fragment in dpb11-1 mutants. (A) YHM011 (DPB11-9myc) and YHM01 (POL2-3HA) cells arrested by α-factor were released to YPD medium without HU and cultured at 16°C (−HU) or to YEPR medium supplemented with HU and cultured at 25°C (+HU) (time = 0 min). The DNA from cells taken at 75 min was subjected to PCR amplification as outlined in the legend to Fig. 3. (B) YHM01 (POL2-3HA), YHM11 (POL2-3HA dpb11-1), and YHM21 (POL2-3HA rad53-1) cells were arrested by α-factor, released in YEPR medium supplemented with 0.2 M HU, and cultured at 25°C. PCR was performed as described in the legend to Fig. 3.