DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links.
DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells.
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Markus
M
Räschle
Ubiquitin Signaling Group, Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Godelieve
G
Smeenk
Ubiquitin Signaling Group, Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Rebecca K
RK
Hansen
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Tikira
T
Temu
Ubiquitin Signaling Group, Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Yasuyoshi
Y
Oka
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Marco Y
MY
Hein
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Nagarjuna
N
Nagaraj
Howard Hughes Medical Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
David T
DT
Long
Howard Hughes Medical Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Johannes C
JC
Walter
Institute of Genetics, University of Cologne, 50674 Cologne, Germany.
Kay
K
Hofmann
Maintenance of Genome Stability Group, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Zuzana
Z
Storchova
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Jürgen
J
Cox
Ubiquitin Signaling Group, Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark. simon.bekker-jensen@cpr.ku.dk niels.mailand@cpr.ku.dk mmann@biochem.mpg.de.
Simon
S
Bekker-Jensen
Ubiquitin Signaling Group, Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark. simon.bekker-jensen@cpr.ku.dk niels.mailand@cpr.ku.dk mmann@biochem.mpg.de.
Niels
N
Mailand
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. Novo Nordisk Foundation Center for Protein Research, Proteomics Program, University of Copenhagen, DK-2200 Copenhagen, Denmark. simon.bekker-jensen@cpr.ku.dk niels.mailand@cpr.ku.dk mmann@biochem.mpg.de.
Matthias
M
Mann
D000818:Animals; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D040901:Proteomics / Q000379:methods; D016601:RNA-Binding Proteins / Q000378:metabolism; D014981:Xenopus
D002843:Chromatin; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; D045643:DNA Repair Enzymes