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. 2004 Apr 1;18(7):769-81.
doi: 10.1101/gad.1173204.

The NAD(+)-dependent Sir2p histone deacetylase is a negative regulator of chromosomal DNA replication

Donald L Pappas Jr  1 , Ryan Frisch, Michael Weinreich
Affiliations

The NAD(+)-dependent Sir2p histone deacetylase is a negative regulator of chromosomal DNA replication

Donald L Pappas Jr et al. Genes Dev. .

Abstract

The establishment of DNA synthesis during the S phase is a multistep process that occurs in several stages beginning in late mitosis. The first step is the formation of a large prereplicative complex (pre-RC) at individual replication origins and occurs during exit from mitosis and entry into G1 phase. To better understand the genetic requirements for pre-RC formation, we selected chromosomal suppressors of a temperature-sensitive cdc6-4 mutant defective for pre-RC assembly. Loss-of-function mutations in the chromatin-modifying genes SIR2, and to a lesser extent in SIR3 and SIR4, suppressed the cdc6-4 temperature-sensitive lethality. This suppression was independent of the well-known silencing roles for the SIR proteins at the HM loci, at telomeres, or at the rDNA locus. A deletion of SIR2 uniquely rescued both the DNA synthesis defect of the cdc6-4 mutant and its severe plasmid instability phenotype for many origins. A SIR2 deletion suppressed additional initiation mutants affecting pre-RC assembly but not mutants that act subsequently. These findings suggest that Sir2p negatively regulates the initiation of DNA replication through a novel mechanism and reveal another connection between proteins that initiate DNA synthesis and those that establish silent heterochromatin in budding yeast.

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Figures

Figure 1.
Figure 1.
Tenfold serial dilutions of strains were spotted onto plates and incubated at 25°C (3 d) and 37°C (2 d). (A) Deletion of SIR2-4 suppresses the temperature-sensitivity of cdc6-4. M138 (W303-1A), M386 (cdc6-4), M636 (cdc6-4 orc1ΔN6–235), M638 (cdc6-4 sir1Δ), M922 (cdc6-4 sir2Δ), M971 (cdc6-4 sir3Δ), and M974 (cdc6-4 sir4Δ). (B) Simultaneous MATa and MATα expression does not suppress cdc6-4. M138 (WT), M386 (MATa cdc6-4), M599 (MATα cdc6–4), M1101 (MATa/MATα cdc6-4/cdc6-4), M1102 (MATα cdc6-4 hmra-ss*), and M576 (MATα cdc6–4[2×]). (C) Disruption of telomeric silencing does not suppress cdc6-4. M1020 (WT, VIIL::URA3-tel), M1021 (cdc6-4 VIIL::URA3-tel), AJL369-4d (rap1-17 VIIL::URA3-tel), M1010 (cdc6-4 rap1-17 VIIL::URA3-tel). (D) Loss of Sir2p deacetylase activity suppresses cdc6-4. M138 (WT), M386 (cdc6-4), M795 (sir2-N345A), and M1100 (cdc6-4 sir2-N345A). (E) Loss of Sir2p rDNA localization does not suppress cdc6-4. M138 (WT), M386 (cdc6-4), M1117 (sir2-87), M1118 (cdc6-4 sir2-87), M1155 (sir2-86), M1164 (cdc6-4 sir2-86), M1156 (sir2-88), and M1166 (cdc6-4 sir2-88).
Figure 2.
Figure 2.
Deletion of SIR2 does not alter the abundance of the wild-type or Cdc6-4 proteins. (A) 12CA5 Western blot for Cdc6p from asynchronous total cell extracts of W303-1A (untagged), M276 (3HA-CDC6), and M1065 (3HA-CDC6 sir2Δ). (B) 12CA5 Western blot of Cdc6-4p and FACS samples from asynchronous cells and cells completely arrested in G2/M phase using 15 μg/mL nocodazole or in G1 phase using 10 μg/mL α-factor; W303-1A (untagged), M1257 (3HA-cdc6-4), M1274 (3HA-cdc6-4 sir2Δ), M1299 (3HA-cdc6-4 sir2Δ hmlΔ). (C) Deletion of SIR1, SIR2, or SIR3 does not suppress the cdc6-1 temperature sensitivity. W303-1A (WT), M379 (cdc6-1), M1103 (cdc6-1 sir1Δ), M1104 (cdc6-1 sir2Δ), M1105 (cdc6-1 sir3Δ).
Figure 3.
Figure 3.
Deletion of SIR2 promotes entry into S phase in the cdc6-4 mutant. M138 (WT), M386 (cdc6-4), and M940 (cdc6-4 sir2Δ hmlΔ) were arrested with α-factor for 3 h at 25°C, shifted for 1 h to 37°C,andthenreleasedfrom thearrestinto fresh YPD medium at 37°C. Cell cycle progression was monitored by flow cytometry as described (Weinreich et al. 1999).
Figure 4.
Figure 4.
(A) Deletion of SIR2 can partially rescue additional pre-RC mutants. M138 (WT), M198 (orc5-1), M1096 (orc5-1 sir2Δ), M359 (mcm2-1), M1097 (mcm2-1 sir2Δ), M444 (cdc7-1), M1098 (cdc7-1 sir2Δ), M354 (cdc17-1), and M1099 (cdc17-1 sir2Δ). (B) Deletion of the histone deacetylases RPD3 or HDA1 will not suppress cdc6-4. M138 (WT), M386 (cdc6-4), WJY140 (rpd3Δ), M1080 (cdc6-4 rpd3Δ), M1161 (hda1Δ), and M1174 (cdc6-4 hda1Δ).
Figure 5.
Figure 5.
Deletion of SIR2 rescues the plasmid instability defect conferred by cdc6-4 at certain origins and improves plasmid stability in wild-type cells. Values were determined as in Dani and Zakian (1983) and are reported are percentage plasmid loss rate per generation in nonselective medium at 25°C. They represent the average of at least four measurements. The ARS501 plasmid is pR151 (Ferguson et al. 1991), ARSH4 is pRS416, and the remaining plasmids have ∼300–500-bp origin fragments replacing the ARS1 origin on pARS1-WT (Marahrens and Stillman 1992).
Figure 6.
Figure 6.
Deletion of SIR2 restores Mcm2p loading at origins. ChIP assays were performed as described in Materials and Methods using cells grown in YPD medium to mid-log phase, arrested in G2/M with 15 μg/mL nocodazole, and then released into YPD medium containing 5 μg/mL α-factor for 60, 90, or 120 min, so that 95% of cells had a 1C DNA content as determined by flow cytometry. W303-1A (WT), M386 (cdc6-4), and M940 (cdc6-4 sir2Δ hmlΔ). A representative input DNA PCR sample is shown for each origin examined.
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