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

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support SIMILAR CONTENT BEING VIEWED BY OTHERS MECHANISM OF AUTO-INHIBITION AND ACTIVATION OF MEC1ATR CHECKPOINT KINASE Article 09 November 2020 MITOTIC DNA SYNTHESIS IN RESPONSE TO

REPLICATION STRESS REQUIRES THE SEQUENTIAL ACTION OF DNA POLYMERASES ZETA AND DELTA IN HUMAN CELLS Article Open access 09 February 2023 SPOP MUTATION INDUCES REPLICATION OVER-FIRING BY

IMPAIRING GEMININ UBIQUITINATION AND TRIGGERS REPLICATION CATASTROPHE UPON ATR INHIBITION Article Open access 01 October 2021 REFERENCES * Painter, R. B. & Young, B. R. Radiosensitivity

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proteomics. _J. Proteome Res._ 1, 21–26 (2002) Article  CAS  Google Scholar  Download references 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. AUTHOR INFORMATION Author notes * Zophonias O. Jonsson Present address: Present address: Institute of Biology, University of Iceland, Sturlugata 7,101-Reykjavik,

Iceland., AUTHORS AND AFFILIATIONS * Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94158-9001, USA, Jaime Lopez-Mosqueda, 

Nancy L. Maas, Lisa G. DeFazio-Eli & David P. Toczyski * Department of Biological Chemistry, University of California, Los Angeles, 615 Charles E. Young Dr. South BSRB 377A, Los Angeles,

California 90095, USA, Zophonias O. Jonsson & James Wohlschlegel Authors * Jaime Lopez-Mosqueda View author publications You can also search for this author inPubMed Google Scholar *

Nancy L. Maas View author publications You can also search for this author inPubMed Google Scholar * Zophonias O. Jonsson View author publications You can also search for this author

inPubMed Google Scholar * Lisa G. DeFazio-Eli View author publications You can also search for this author inPubMed Google Scholar * James Wohlschlegel View author publications You can also

search for this author inPubMed Google Scholar * David P. Toczyski View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS 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. CORRESPONDING AUTHOR Correspondence to David P. Toczyski. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests.

SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION This file contains Supplementary Tables 1 – 3 and Supplementary Figures 1-12 with legends. (PDF 4169 kb) POWERPOINT SLIDES POWERPOINT

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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 Download citation * Received: 28 July 2009 * Accepted: 22 July 2010 * Issue Date: 23 September 2010 * DOI: https://doi.org/10.1038/nature09377 SHARE THIS

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