The NAD(+)-dependent Sir2p histone deacetylase is a negative regulator of chromosomal DNA replication.
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.
Pappas
Donald L
DL
Laboratory of Chromosome Replication, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA.
Frisch
Ryan
R
Weinreich
Michael
M
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D015825:Chromosomes, Fungal / Q000235:genetics*; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism*; D026341:Minichromosome Maintenance 1 Protein / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D056565:Sirtuin 2; D037761:Sirtuins / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics; D013696:Temperature
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D026341:Minichromosome Maintenance 1 Protein; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D009243:NAD; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases