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Damage-induced phosphorylation of Sld3 is important to block late origin firing

Abstract

Origins of replication are activated throughout the S phase of the cell cycle 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 telangiectasia mutated (ATM) kinase and elicit radioresistant DNA synthesis1,2,3 after γ-irradiation2. This effect is conserved in budding yeast, as yeast cells lacking the related kinase Mec1 (ATM and Rad3-related (ATR in humans)) 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 in humans)5,6. However, the Rad53 substrate(s) whose phosphorylation is required to mediate this function has 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 in Saccharomyces cerevisiae. Upon exposure to DNA-damaging agents, cells expressing non-phosphorylatable alleles of SLD3 and DBF4 (SLD3-m25 and dbf4-m25, respectively) proceed through the 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 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 indicate 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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Figure 1: Rad53-dependent phosphorylation of Sld3.
Figure 2: SLD3-m25 and dbf4-m25 cells are intra-S-phase checkpoint defective.
Figure 3: Inappropriate firing of late origins in the presence of replication inhibitors.
Figure 4: Inappropriate firing of late origins elicits DNA damage.

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Acknowledgements

We would like to thank members of the Toczyski, Morgan, Li and O’Farrell laboratories for discussions. We also thank D. Morgan, G. Narlikar and J. Li for intellectual contributions, J. Benanti and M. Downey for critical reading of this manuscript, and S. Makovets and M. Hoang for assistance with two-dimensional DNA gels. We thank H. Araki for strains, R. Sclafani for plasmids and J. Diffley for communicating results before publication. We are especially grateful to N. Lopez for help with GFP foci and colony quantification. Funding was provided by a Ford Foundation Pre-Doctoral Diversity Fellowship and a National Institutes of Health grant GM059691.

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L.G.D.-E. identified Sld3 in initial proteomic screen. Z.O.J. and J.W. performed mass spectrometry analysis. J.L.-M. performed all other experiments with assistance from N.L.M. J.L.M. and D.P.T. designed experiments and wrote the manuscript.

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Correspondence to David P. Toczyski.

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Lopez-Mosqueda, J., Maas, N., Jonsson, Z. et al. Damage-induced phosphorylation of Sld3 is important to block late origin firing. Nature 467, 479–483 (2010). https://doi.org/10.1038/nature09377

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