CtIP-mediated resection is essential for viability and can operate independently of BRCA1.
Homologous recombination (HR) is initiated by DNA end resection, a process in which stretches of single-strand DNA (ssDNA) are generated and used for homology search. Factors implicated in resection include nucleases MRE11, EXO1, and DNA2, which process DNA ends into 3' ssDNA overhangs; helicases such as BLM, which unwind DNA; and other proteins such as BRCA1 and CtIP whose functions remain unclear. CDK-mediated phosphorylation of CtIP on T847 is required to promote resection, whereas CDK-dependent phosphorylation of CtIP-S327 is required for interaction with BRCA1. Here, we provide evidence that CtIP functions independently of BRCA1 in promoting DSB end resection. First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions. Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP(-/-) mice. Third, the increased resection afforded by loss of 53BP1 and the rescue of BRCA1-deficiency depend on CtIP but not EXO1. Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E). Thus, in contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development.
Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Federica
F
Polato
Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Elsa
E
Callen
Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Nancy
N
Wong
Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Robert
R
Faryabi
Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Samuel
S
Bunting
Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Hua-Tang
HT
Chen
Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Marina
M
Kozak
Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Michael J
MJ
Kruhlak
Institute for Cancer Genetics, Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY 10032.
Colleen R
CR
Reczek
Department of Biological Chemistry, School of Medicine, University of California, Irvine, CA 92697.
Wen-Hwa
WH
Lee
Ohio State University Wexner Medical Center, Columbus, OH 43210.
Thomas
T
Ludwig
Institute for Cancer Genetics, Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY 10032.
Richard
R
Baer
Science Applications International Corporation-Frederick National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21704.
Lionel
L
Feigenbaum
The Wellcome Trust and Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, England, UK The Wellcome Trust and Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, England, UK The Wellcome Trust Sanger Institute, Hinxton CB10 1SA, England, UK.
Stephen
S
Jackson
Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 andre_nussenzweig@nih.gov.
André
A
Nussenzweig
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019313:BRCA1 Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D002868:Chromosomal Proteins, Non-Histone / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D042822:Genomic Instability; D059765:Homologous Recombination; D015151:Immunoblotting; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D009154:Mutation; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding; D000071857:Tumor Suppressor p53-Binding Protein 1
D019313:BRCA1 Protein; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases; D000071857:Tumor Suppressor p53-Binding Protein 1; C501070:RBBP8 protein, mouse; C498302:Trp53bp1 protein, mouse