pubmed.ncbi.nlm.nih.gov

DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae - PubMed

  • ️Fri Jan 01 1999

DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae

H Wang et al. Proc Natl Acad Sci U S A. 1999.

Abstract

In addition to DNA polymerase complexes, DNA replication requires the coordinate action of a series of proteins, including regulators Cdc28/Clb and Dbf4/Cdc7 kinases, Orcs, Mcms, Cdc6, Cdc45, and Dpb11. Of these, Dpb11, an essential BRCT repeat protein, has remained particularly enigmatic. The Schizosaccharomyces pombe homolog of DPB11, cut5, has been implicated in the DNA replication checkpoint as has the POL2 gene with which DPB11 genetically interacts. Here we describe a gene, DRC1, isolated as a dosage suppressor of dpb11-1. DRC1 is an essential cell cycle-regulated gene required for DNA replication. We show that both Dpb11 and Drc1 are required for the S-phase checkpoint, including the proper activation of the Rad53 kinase in response to DNA damage and replication blocks. Dpb11 is the second BRCT-repeat protein shown to control Rad53 function, possibly indicating a general function for this class of proteins. DRC1 and DPB11 show synthetic lethality and reciprocal dosage suppression. The Drc1 and Dpb11 proteins physically associate and function together to coordinate DNA replication and the cell cycle.

PubMed Disclaimer

Figures

Figure 1
Figure 1

The S-phase checkpoint is deficient in dpb11–1 mutant. (A–C) Wild-type (Y791) and dpb11–1 (Y792) cells were synchronized by α-factor at 24°C. After G1 arrest, cultures were shifted to 36°C for 45 min and divided into two, and equal portions were released from the block into SC-Ura or SC-Ura containing 0.2 M HU. At regular intervals after release, aliquots were withdrawn to examine DNA content, nuclear and spindle morphology. (A) FACS profile shows the DNA content of wild-type and dpb11–1 cells after release from G1 into medium with or without 0.2 M HU. Time indicates minutes after release. (Lower) Asynchronous cells untreated with HU at 24°C, which are included as a reference. (B) Photomicrographs of wild-type and dpb11–1 cells at 120 min after release from α-factor into SC-Ura with 0.2 M HU at 36°C. Nuclear morphology was visualized with 4′,6-diamidino-2-phenylindole, and microtubule morphology was visualized by indirect immunofluorescence using anti-α-tubulin antibody. (C) Kinetics of spindle elongation in the presence or absence of HU. (D) Y791 and Y792 were grown in SC-Ura, synchronized in G1 by α-factor, then prewarmed at 34°C for 1 hr before release at 34°C into media lacking α-factor. At 30 min after release, when most of the cells enter S phase, HU (0.2 M final) or MMS (0.1% final) was added to block DNA replication or damage DNA. An hour later (90 min after release), protein extracts were prepared, fractionated by SDS/PAGE, and immunoblotted with antibodies to Rad53 (2).

Figure 2
Figure 2

(A) High copy of DRC1 rescues the growth defect of dpb11–1. dpb11–1 containing either pRS326 (2 μ URA3) or pHW21(2 μ URA3 DRC1) were struck out on SC-Ura plates and incubated at 24°C and 34°C. (B) Conservation between Drc1 and a related protein from S. pombe. Identities are shown as black boxes and similarities as shaded boxes. (C) High-copy DRC1 suppresses the lethality of pol2 mutants. TC102–2D (POL2), TC102–2–11 (pol2–11), TC102–12 (pol2–12) (19), YHA300 (POL2), and YHA301 (pol2–18) (20) strains were transformed with either pRS326 (−) or pHW21(DRC1) (+). Cultures of those transformants were grown in SC-Ura medium at 24°, then serial dilutions of 105, 104, 103, 102, and 10 cells were spotted onto SC-Ura plates and incubated at the indicated temperatures.

Figure 3
Figure 3

(A) Cell cycle regulation of DRC1. Cultures of wild-type (Y300) cells were synchronized with α-factor and released into YPD at 30°C. Samples for RNA isolation were collected at 10 min, and Northern blot analysis was performed by using DRC1, RNR1, and ACT1 as probes. (B and C) drc1–1 mutants are defective in DNA synthesis at the restrictive temperature. Cells from both W303 ρ0 (wild type) (Y794) and an isogenic drc1–1 derivative (Y795) were arrested with α-factor at 24°C, then shifted to 37°C for 1 hr before releasing from the α-factor block into YPD at 37°C. Aliquots were removed at 30 min intervals for analysis of the budding profile (B) and DNA content by FACS (C).

Figure 4
Figure 4

drc1–8 mutants are defective for the S-phase checkpoint. (A–D) Wild-type (Y796) and drc1–8 (Y798) cells were synchronized by α-factor at 24°C. After G1 arrest, cultures were divided into two and released from the block into either YPD or YPD containing 0.2 M HU. At regular intervals after release, aliquots were withdrawn to examine DNA content, nuclear and spindle morphology, and cell viability. (A) FACS profile showing the DNA content of wild-type and drc1–8 cells after release from G1 into medium with or without 0.2 M HU. Time indicates minutes after release. (Lower) Untreated asynchronous cells at 24°C and are included as a reference. (B) Photomicrographs of wild-type and drc1–8 cells at 150 min after release from α-factor into YPD with 0.2 M HU. Nuclear morphology was visualized with 4′,6-diamidino-2-phenylindole, and microtubule morphology was visualized by indirect immunofluorescence using anti-α-tubulin antibodies. (C) Kinetics of spindle elongation in the presence or absence of HU. (D) Cell viability was analyzed by scoring colony-forming units on YPD plates at 24°C. (E) Wild-type (Y300), drc1–1 (Y799), and drc1–8 (Y798) cells were synchronized in G1 by α-factor at 24°C. After release for 30 min, HU or MMS was added to block DNA replication block or damage DNA for 1 hr. Protein extracts were prepared, separated by SDS/PAGE, and immunoblotted with antibodies to Rad53.

Figure 5
Figure 5

DPB11 and DRC1 show reciprocal suppression and their proteins physically associate. (A) The Ts phenotype of drc1–1 is suppressed by the overproduction of DPB11. Y799 (drc1–1) cells harboring either an empty vector, pRS326, or DPB11 on a 2-μ plasmid, YEp195DPB11, were struck upon SC-Ura plates and incubated at 24°C, 30°C, and 37°C respectively. (B) Physical interaction between Dpb11 and Drc1 in vitro. GST or GST-Dpb11 bound to glutathione beads purified from baculovirus-infected insect cells were incubated with 35S-methionine-labeled Drc1 made by in vitro translation. Proteins bound to the beads after several washes were resolved by SDS/PAGE (10%) and visualized by autoradiography.

Similar articles

Cited by

References

    1. Elledge S J. Science. 1996;274:1664–1672. - PubMed
    1. Sanchez Y, Desany B A, Jones W J, Liu Q, Wang B, Elledge S J. Science. 1996;271:357–360. - PubMed
    1. Sun Z, Fay D S, Marini F, Foiani M, Stern D F. Genes Dev. 1996;10:395–406. - PubMed
    1. Weinert T. Cell. 1998;94:555–558. - PubMed
    1. Longhese M P, Paciotti V, Fraschini R, Zaccarini R, Plevani P, Lucchini G. EMBO J. 1997;16:5216–5226. - PMC - PubMed

Publication types

MeSH terms

Substances