Damage-Induced Phosphorylation of Sld3 is Important to Block Late Origin Firing

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94158-9001, USA.
Nature (Impact Factor: 41.46). 09/2010; 467(7314):479-83. DOI: 10.1038/nature09377
Source: PubMed


Origins of replication are activated throughout S-phase such that some origins fire early and others fire late to ensure that each chromosome is completely replicated in a timely fashion. However, in response to DNA damage or replication fork stalling, eukaryotic cells block activation of unfired origins. Human cells derived from patients with ataxia telangiectasia are deficient in this process due to the lack of a functional ataxia-telegiectasia mutated (ATM) kinase and elicit Radio-resistant DNA synthesis (RDS)1–3 following γ-irradiation2. This effect is conserved in budding yeast, as yeast cells lacking the related kinase Mec1 (ATR) also fail to inhibit DNA synthesis in the presence of DNA damage4. This intra-S-phase checkpoint actively regulates DNA synthesis by inhibiting the firing of late replicating origins, and this inhibition requires both Mec1 and the downstream checkpoint kinase Rad53 (Chk2)5,6. However, the Rad53 substrate(s) whose phosphorylation is required to mediate this function remained unknown. Here, we show that the replication initiation protein Sld3 is phosphorylated by Rad53, and that this phosphorylation, along with phosphorylation of the Cdc7 kinase regulatory subunit Dbf4, blocks late origin firing. Upon exposure to DNA damaging agents, cells expressing nonphosphorylatable alleles of SLD3 and DBF4 (SLD3-m25 and dbf4-m25, respectively) proceed through S-phase faster than wild-type cells by inappropriately firing late origins of replication. SLD3-m25 dbf4-m25 cells grow poorly in the presence of the replication inhibitor hydroxyurea (HU) and accumulate multiple Rad52 foci. Moreover, SLD3-m25 dbf4-m25 cells are delayed in recovering from transient blocks to replication and subsequently arrest at the DNA damage checkpoint. These data suggest that the intra-S-phase checkpoint functions to block late origin firing in adverse conditions to prevent genomic instability and maximize cell survival.

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Available from: Zophonías O. Jónsson
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    • "Activated Rad53 inhibits the firing of late replication origins (Santocanale and Diffley, 1998), maintains fork stability (Lopes et al., 2001), and regulates replication fork restart (Szyjka et al., 2008). It is known that Rad53 inhibits firing of late replication origins through phosphorylation of Sld3, a protein involved in initiation of DNA replication and Dbf4 (a subunit of Cdc7/Dbf4 kinase) (Lopez-Mosqueda et al., 2010; Zegerman and Diffley, 2010). However, it remains poorly understood how checkpoint kinases maintain fork stability in response to replication stress. "
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    • "Importantly, in case of DNA damage, Rad53 functions to inhibit both CDK-and DDK-dependent activation pathways . Rad53 acts on DDK directly by phosphorylating Dbf4, whereas the CDK pathway is blocked by Rad53-mediated phosphorylation of the essential CDK substrate, Sld3 (Duch et al. 2011; Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010). This regulatory principle inactivates DDK, but allows CDK to remain active during S phase in the presence of DNA damage, which is crucial to prevent relicensing of origins that have already fired. "
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    • "The budding yeast Mec1 and Rad53 are the key protein kinases of the genome integrity checkpoint, a complex genome surveillance mechanism that integrates signals from stalled replication forks and DNA breaks. In response to DNA damage or replication stress, the genome integrity checkpoint helps to maintain and recover stalled replication forks (1–5), blocks the activation of late replication origins (6–8) and, via the downstream kinase Dun1, activates DNA repair proteins (9). One notable checkpoint-activated protein is ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in the biosynthesis of all four deoxyribonucleoside triphosphates (dNTPs) and maintains both their balance and appropriate overall concentrations. "
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