Dataset Information

Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links part 2 of 2: AP-MS DATA (QUBIC)

ABSTRACT: DNA interstrand crosslinks (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. Here we established 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 at ICL-containing chromatin. Among numerous prospective new DNA repair factors we identified SLF1 and SLF2, which form a complex with RAD18 and together define a new pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides the first global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Dr Matthias Mann

PROVIDER: MSV000079577 | MassIVE | Sun Mar 13 12:00:00 GMT 2016

SECONDARY ACCESSION(S): PXD000491

REPOSITORIES: MassIVE

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Publications

DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links.

Räschle Markus M Smeenk Godelieve G Hansen Rebecca K RK Temu Tikira T Oka Yasuyoshi Y Hein Marco Y MY Nagaraj Nagarjuna N Long David T DT Walter Johannes C JC Hofmann Kay K Storchova Zuzana Z Cox Jürgen J Bekker-Jensen Simon S Mailand Niels N Mann Matthias M

Science (New York, N.Y.) 20150430 6234

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 prote ...[more]

PMID: 25931565

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Project description:The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

Dataset Information

Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links part 2 of 2: AP-MS DATA (QUBIC)

Publications

DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links.

Similar Datasets

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