Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are suppressed by deletion of either SIR2 or SIR3. While these and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a SIR2 deletion rescued MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner in wild type (i.e. CDC6) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin–SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 –correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins.

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK-and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5-and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.T HE fidelity of chromosome replication depends on checkpoint mechanisms to stabilize stalled forks, regulate origin activation, and repair DNA damage (Hartwell and Weinert 1989;Bartek et al. 2004;Segurado and Tercero 2009). In response to replication stress, the replication checkpoint maintains replisome stability and prevents late origins from firing, which allows time for DNA repair and the completion of DNA replication prior to chromosome segregation. Incomplete DNA replication or uncoordinated origin firing following DNA damage can result in genomic instability, cancer predisposition, and premature aging (Branzei and Foiani 2010).In the budding yeast Saccharomyces cerevisiae, activation of the checkpoint sensor kinase Mec1 (vertebrate ATR, Ataxia Telangiectasia and Rad3-related) is triggered at stalled forks or sites of DNA damage (Majka et al. 2006;Labib and De Piccoli 2011). Subsequent signal amplification through the Mrc1 or Rad9 adaptors leads to activation of the checkpoint kinase Rad53 (the ortholog of the human tumor suppressor Chk2) (Branzei and Foiani 2009). Rad53 is an integral transducer of various cellular responses to replication stress or DNA damage. Rad53 induces a series of transcriptional responses through MBF-regulated genes (Bastos de Oliveira et al. 2012;Travesa et al. 2012) and also activates the Dun1 kinase, which promotes the expression of ribonucleotide reductase (RNR) subunits and additional DNA repair genes (Huang et al. 1998). In parallel, Rad53 down-regulates the RNR inhibitor Sml1 to increase deoxyribonucleotide levels and facilitate DNA synthesis (Zhao et al. 2001). In response to replication fork stalling, Rad53 prevents the activation of late replication origins by phosphorylating two proteins required for the initiation of DNA replication: Dbf4 and Sld3 (...

25Most active DNA replication origins are found within euchromatin, while origins within 26 heterochromatin are often inactive or inhibited. In yeast, origin activity within 27 heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at 28 some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing 29 view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. 30However, growth defects in yeast mutants compromised for loading the MCM helicase, 31 such as cdc6-4, are robustly suppressed by deletion of either SIR2 or SIR3. While this 32 and other observations indicate that SIR2,3 can have a negative impact on at least some 33 euchromatic origins, the genomic scale of this effect was unknown. It was also unknown 34 whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or 35 was an indirect effect of their previously established roles within heterochromatin. Using 36 both MCM ChIP-Seq and MNase-H4K16ac ChIP-Seq data, we show that a SIR2 37 deletion rescues MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. 38Therefore, Sir2 exhibits a pervasive effect at the majority of euchromatic origins. 39Importantly, in wild type (i.e. CDC6) cells, origin-adjacent nucleosomes were depleted 40 for H4K16 acetylation in a SIR2-dependent manner. In addition, both Sir2 and Sir3 41 directly bound to nucleosomes adjacent to euchromatic origins. The relative levels of 42 each of these molecular hallmarks of yeast heterochromatin -SIR2-dependent H4K16 43 hypoacetylation, Sir2, and Sir3 -correlated with how strongly a SIR2 deletion 44 suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and 45 H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that 46 map to a surface of the nucleosome important for Sir3 binding. We conclude that 47 heterochromatin proteins directly bind euchromatic DNA replication origins and modify 48 their local chromatin environment. 49 50 3 AUTHOR SUMMARY 51When a cell divides, it must copy or "replicate" its DNA. DNA replication starts at 52 chromosomal regions called origins when a collection of replication proteins gains local 53 access to unwind the two strands. Chromosomal DNA is packaged into a protein-DNA 54 complex called chromatin and there are two major structurally and functionally distinct 55 types. Euchromatin allows DNA replication proteins to access origin DNA, while 56 heterochromatin inhibits their access. The prevalent view has been that the 57 heterochromatin proteins required to inhibit origins are confined to heterochromatin. In 58 this study, the conserved heterochromatin proteins, Sir2 and Sir3, are shown to both 59 physically and functionally associate with the majority of origins in euchromatin. This 60 raises important new questions about the chromosomal targets of heterochromatin 61 proteins, and how and why the majority of origins exist within a potentially repressive 62 chromatin structure. 63 64 replic...