Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks.
The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase-specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605;
Nicholas A
NA
Willis
Department of Genetics, Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; The Laboratory of Medical Genetics, Harbin Medical University, Harbin, China, 150081;
Chunshui
C
Zhou
Department of Genetics, Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA 02114;
Andrew E H
AE
Elia
Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, BN1 9RQ, United Kingdom;
Johanne M
JM
Murray
Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, BN1 9RQ, United Kingdom;
Antony M
AM
Carr
Department of Genetics, Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115 selledge@genetics.med.harvard.edu nick.rhind@umassmed.edu.
Stephen J
SJ
Elledge
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605; selledge@genetics.med.harvard.edu nick.rhind@umassmed.edu.
Nicholas
N
Rhind
D064447:Checkpoint Kinase 2 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D042822:Genomic Instability / Q000187:drug effects; D008741:Methyl Methanesulfonate / Q000633:toxicity; D016196:S Phase / Q000187:drug effects; D012568:Schizosaccharomyces / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism
D029702:Schizosaccharomyces pombe Proteins; D008741:Methyl Methanesulfonate; D064447:Checkpoint Kinase 2; C578636:Cds1 protein, S pombe; C456840:rad3 protein, S pombe
Cds1; DDR; DNA damage; Rad3; checkpoint