Dataset Information

Human HEK293T cells LC-MS/MS (Gel-based proteomics)

ABSTRACT: Fanconi Anemia (FA) pathway is essential for the repair of inter-strand crosslink (ICLs). ICL induces stalled replication forks and triggers activation of FA pathway for efficient ICL repair. Given that stalled replication fork is proximal to ICLs sites, fork-associated proteins may likely coordinate with FA factors to rapidly sense ICLs for activation of FA signaling. We will use LC-MS/MS to identify such proteins that participated in the ICL repairs.

INSTRUMENT(S): LTQ

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture, Kidney

SUBMITTER: YI ZHANG

LAB HEAD: Wenge Zhu

PROVIDER: PXD028246 | Pride | 2023-05-10

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	QE_Sample1_F1_R1.raw	Raw
	QE_Sample2_F1_R1.raw	Raw
	Samplelist.xlsx	Xlsx

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Publications

And-1 Coordinates with the FANCM Complex to Regulate Fanconi Anemia Signaling and Cisplatin Resistance.

Zhang Yi Y Li Jing J Zhou Yuan Y Li Zhuqing Z Peng Changmin C Pei Huadong H Zhu Wenge W

Cancer research 20220901 18

The Fanconi anemia (FA) pathway is essential for repairing DNA interstrand crosslinks (ICL). ICLs induce stalled DNA replication forks and trigger activation of the FA pathway by promoting recruitment of the FANCM/FAAP24/MHF complex to ICL sites. Given that stalled replication forks are proximal to ICL sites, fork-associated proteins may coordinate with FA factors to rapidly sense ICLs for activation of FA signaling. Here we report that And-1, a replisome protein, is critical for activation of t ...[more]

PMID: 35867033

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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.