A role for a replicator dominance mechanism in silencing.

The role of the natural HMR-E silencer in modulating replication initiation and silencing by the origin recognition complex (ORC) was examined. When natural HMR-E was the only silencer controlling HMR, the silencer's ORC-binding site (ACS) was dispensable for replication initiation but essential for silencing, indicating that a non-silencer chromosomal replicator(s) existed in close proximity to the silencer. Further analysis revealed that regions flanking both sides of HMR-E contained replicators. In contrast to replication initiation by the intact silencer, initiation by the non-silencer replicator(s) was abolished in an orc2-1 mutant, indicating that these replicators were extremely sensitive to defects in ORC. Remarkably, the activity of one of the non-silencer replicators correlated with reduced silencing; inactivation of these replicators caused by either the orc2-1 mutation or the deletion of flanking sequences enhanced silencing. These data were consistent with a role for the ORC bound to the HMR-E silencer ACS in suppressing the function of neighboring ORC molecules capable of inhibiting silencing, and indicated that differences in ORC-binding sites within HMR itself had profound effects on ORC function. Moreover, replication initiation by natural HMR-E was inefficient, suggesting that closely spaced replicators within HMR contributed to an inhibition of replication initiation.     

Proliferating cell nuclear antigen (pol30) mutations suppress cdc44 mutations and identify potential regions of interaction between the two encoded proteins.

In addition to its role as a processivity factor in DNA replication, proliferating cell nuclear antigen (PCNA) may function in the regulation of cell cycle progression. We present genetic evidence that PCNA interacts with the gene product of CDC44, an essential nucleotide-binding protein that encodes the large subunit of yeast replication factor C (K. Fien and B. Stillman, personal communication). Mutations in POL30 (PCNA) suppress cold-sensitive alleles of cdc44 that contain mutations in or near nucleotide-binding consensus domains, but they do not suppress a null allele. Thus, it appears that PCNA interacts with Cdc44p but cannot substitute for its function. pol30 mutations suppress additional phenotypes of cdc44 mutations, including the cold sensitivity that they were selected to suppress. This observation suggests an intimate association between PCNA and Cdc44p. Each of five independent pol30 mutants contains a unique single mutation that maps to a localized region on one face of the predicted three-dimensional structure of PCNA. This face identifies a region likely to be important for functional interaction between the CDC44 and POL30 gene products.     

Proliferating cell nuclear antigen and ASF1 modulate silent chromatin in Saccharomyces cerevisiae via lysine 56 on histone H3

The formation and stability of epigenetically regulated chromatin is influenced by DNA replication and factors that modulate post-translational modifications on histones. Here we describe evidence that PCNA can affect silencing in Saccharomyces cerevisiae by facilitating deposition of H3 K56ac onto chromosomes. We propose that PCNA participates in this process through a pathway that includes replication factor C, the chromatin assembly factor Asf1p, and the K56-specific acetyltransferase Rtt109p. We show that mutation of POL30 or loss of K56-acetylation in rtt109 and histone H3 mutants enhances silencing at the crippled HMR locus HMRae via restoring Sir binding and that pol30 mutants with silencing phenotypes have reduced levels of H3 K56ac. Although loss of acetylation on H3 K56 was generally compatible with silencing, mutations at this residue also led to defects in silencing an ADE2 reporter at HMR and abolished silencing when combined with cac1 or pol30-8. These silencing phenotypes are analogous to those in asf1 mutants or pol30-6 and pol30-79 mutants with defects in ASF1-dependent pathways. On the basis of these findings, we propose that mutations in DNA replication factors alter acetylation of H3 K56. We show that this defect, in turn, contributes to misregulation of epigenetic processes as well as of cellular responses to DNA damage.     

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.     

Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene.

The budding yeast Saccharomyces cerevisiae is proving to be an useful and accurate model for eukaryotic DNA replication. It contains both DNA polymerase alpha (I) and delta (III). Recently, proliferating cell nuclear antigen (PCNA), which in mammalian cells is an auxiliary subunit of DNA polymerase delta and is essential for in vitro leading strand SV40 DNA replication, was purified from yeast. We have now cloned the gene for yeast PCNA (POL30). The gene codes for an essential protein of 29 kDa, which shows 35% homology with human PCNA. Cell cycle expression studies, using synchronized cells, show that expression of both the PCNA (POL30) and the DNA polymerase delta (POL3, or CDC2) genes of yeast are regulated in an identical fashion to that of the DNA polymerase alpha (POL1) gene. Thus, steady state mRNA levels increase 10-100-fold in late G1 phase, peak in early S-phase, and decrease to low levels in late S-phase. In addition, in meiosis mRNA levels increase prior to initiation of premeiotic DNA synthesis.     

The Role of Sas2, an Acetyltransferase Homologue of Saccharomyces Cerevisiae, in Silencing and Orc Function

Silencing at the cryptic mating-type loci HML and HMR of Saccharomyces cerevisiae requires regulatory sites called silencers. Mutations in the Rap1 and Abf1 binding sites of the HMR-E silencer (HMRa-e**) cause the silencer to be nonfunctional, and hence, cause derepression of HMR. Here, we have isolated and characterized mutations in SAS2 as second-site suppressors of the silencing defect of HMRa-e**. Silencing conferred by the removal of SAS2 (sas2Δ) depended upon the integrity of the ARS consensus sequence of the HMR-E silencer, thus arguing for an involvement of the origin recognition complex (ORC). Restoration of silencing by sas2Δ required ORC2 and ORC5, but not SIR1 or RAP1. Furthermore, sas2Δ suppressed the temperature sensitivity, but not the silencing defect of orc2-1 and orc5-1. Moreover, sas2Δ had opposing effects on silencing of HML and HMR. The putative Sas2 protein bears similarities to known protein acetyltransferases. Several models for the role of Sas2 in silencing are discussed.     

CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.     

CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.     

CDC7/DBF4 functions in the translesion synthesis branch of the RAD6 epistasis group in Saccharomyces cerevisiae

CDC7 and DBF4 encode the essential Cdc7-Dbf4 protein kinase required for DNA replication in eukaryotes from yeast to human. Cdc7-Dbf4 is also required for DNA damage-induced mutagenesis, one of several postreplicational DNA damage tolerance mechanisms mediated by the RAD6 epistasis group. Several genes have been determined to function in separate branches within this group, including RAD5, REV3/REV7 (Pol zeta), RAD30 (Pol eta), and POL30 (PCNA). An extensive genetic analysis of the interactions between CDC7 and REV3, RAD30, RAD5, or POL30 in response to DNA damage was done to determine its role in the RAD6 pathway. CDC7, RAD5, POL30, and RAD30 were found to constitute four separate branches of the RAD6 epistasis group in response to UV and MMS exposure. CDC7 is also shown to function separately from REV3 in response to MMS. However, they belong in the same pathway in response to UV. We propose that the Cdc7-Dbf4 kinase associates with components of the translesion synthesis pathway and that this interaction is dependent upon the type of DNA damage. Finally, activation of the DNA damage checkpoint and the resulting cell cycle delay is intact in cdc7Delta mcm5-bob1 cells, suggesting a direct role for CDC7 in DNA repair/damage tolerance.     

Tolerance of Sir1p/origin recognition complex-dependent silencing for enhanced origin firing at HMRa

The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site.     

Dominant mutations in three different subunits of replication factor C suppress replication defects in yeast PCNA mutants

To identify proteins that interact with the yeast proliferating cell nuclear antigen (PCNA), we used a genetic approach to isolate mutations that compensate for the defects in cold-sensitive (Cs(-)) mutants of yeast PCNA (POL30). Because the cocrystal structure of human PCNA and a p21(WAF1/CIP1) peptide shows that the interdomain region of PCNA is a site of p21 interaction, we specifically looked for new mutations that suppress mutations in the equivalent region of yeast PCNA. In independent screens using three different Cs(-) mutants, we identified spontaneously arising dominant suppressor mutations in the RFC3 gene. In addition, dominant suppressor mutations were identified in the RFC1 and RFC2 genes using a single pol30 mutant. An intimate association between PCNA and RFC1p, RFC2p, and RFC3p is suggested by the allele-restricted suppression of 10 different pol30 alleles by the RFC suppressors. RFC1, RFC2, and RFC3 encode three of the five subunits of the replication factor C complex, which is required to load PCNA onto DNA in reconstituted DNA replication reactions. Genomic sequencing reveals a common region in RFC1p, RFC2p, and RFC3p that is important for the functional interaction with PCNA. Biochemical analysis of the wild type and mutant PCNA and RFC3 proteins shows that mutant RFC3p enhances the production of long DNA products in pol delta-dependent DNA synthesis, which is consistent with an increase in processivity.     

Cell cycle requirements in assembling silent chromatin in Saccharomyces cerevisiae

The establishment of silencing at the silent mating-type locus, HMR, in Saccharomyces cerevisiae requires that yeast pass through S phase of the cell cycle, yet requires neither the initiation of DNA replication at the locus destined to become silenced nor the passage of a replication fork through that locus. We tested whether this S-phase requirement reflects a window within the cell cycle permissive for recruitment of Sir proteins to HMR. The S-phase-restricted event necessary for silencing occurred after recruitment of Sir proteins to HMR. Moreover, cells arrested in early S phase formed silent chromatin at HMR, provided HMR was on a nonreplicating template. Replicating templates required a later step for silencing. These results provide temporal resolution of discrete steps in the formation of silent chromatin and suggest that more than one cell cycle-regulated event may be necessary for the establishment of silencing.