Homologs of the chromatin-bound yeast silent information regulator 2 (SIR2) protein are found in organisms from all biological kingdoms. SIR2 itself was originally discovered to influence mating-type control in haploid cells by locus-specific transcriptional silencing. Since then, SIR2 and its homologs have been suggested to play additional roles in suppression of recombination, chromosomal stability, metabolic regulation, meiosis, and aging. Considering the far-ranging nature of these functions, a major experimental goal has been to understand the molecular mechanism(s) by which this family of proteins acts. We report here that members of the SIR2 family catalyze an NAD-nicotinamide exchange reaction that requires the presence of acetylated lysines such as those found in the N termini of histones. Significantly, these enzymes also catalyze histone deacetylation in a reaction that absolutely requires NAD, thereby distinguishing them from previously characterized deacetylases. The enzymes are active on histone substrates that have been acetylated by both chromatin assemblylinked and transcription-related acetyltransferases. Contrary to a recent report, we find no evidence that these proteins ADPribosylate histones. Discovery of an intrinsic deacetylation activity for the conserved SIR2 family provides a mechanism for modifying histones and other proteins to regulate transcription and diverse biological processes.Y east silent information regulator 2 (SIR2) protein functions in transcriptional silencing of the silent mating loci, telomeres, and rDNA (1-3). It is found in a chromatin-bound complex with SIR3 and SIR4 at the silent mating loci and telomeres, and in a different complex at rDNA (4-6). Four additional SIR2 homologs exist in yeast (HST1-4), and related proteins are found from archaeabacteria to eubacteria to mammals (7). Until recently, very little was known about the in vivo activity of this family of proteins. An important breakthrough came with the identification of the Salmonella typhimurium CobB protein as a SIR2 homolog (8). CobB can partially fulfill the requirement for CobT in vitamin B 12 synthesis. Because CobT protein was known to transfer ribose 5Ј-phosphate from nicotinic acid mononucleotide to a precursor of vitamin B 12 , it prompted tests of Sir2-like proteins for phosphoribosyltransferase activity. Indeed, Frye (9) found that Escherichia coli CobB had NAD-dependent ADP-ribosyltransferase activity. He also reported that both CobB and a human SIR2-like protein could transfer radioactivity from [ 32 P]NAD to albumin. Very recently, another group (10) reported that yeast SIR2 can ADP-ribosylate itself as well as histones and albumin.Here we show that members of the SIR2 family of enzymes catalyze an NAD-nicotinamide exchange reaction that requires the presence of acetylated lysines such as are found in the N termini of histones. Furthermore, these enzymes also catalyze histone deacetylation in a reaction that absolutely depends on NAD, thereby distinguishing them from previously known deacet...

Summary Proper eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an in vitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) initially drives origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Corresponding, in vivo studies demonstrate that DDK drives early-firing origin recruitment of Cdc45 before S-CDK activation. Upon activation of S-phase cyclin-dependent kinases (S-CDK), a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Investigation of DNA polymerase recruitment showed that Mcm10 and DNA unwinding both were critical for recruitment of lagging but not leading strand DNA polymerases. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which leading strand DNA polymerases are recruited prior to origin DNA unwinding and RNA primer synthesis.

Failure to reactivate either stalled or collapsed replication forks is a source of genomic instability in both prokaryotes and eukaryotes. In prokaryotes, dedicated fork repair systems that involve both recombination and replication proteins have been identified genetically and characterized biochemically. Replication conflicts are solved through several pathways, some of which require recombination and some of which operate directly at the stalled fork. Some recent biochemical observations support models of direct fork repair in which the removal of the blocking template lesion is not always required for replication restart.