Dataset Information

Improved detection of DNA replication fork-associated proteins via iPOND2-DRIPPER

ABSTRACT: Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of di-glycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components, and nuclear cytoskeleton networks.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Ya-Chu Chang

LAB HEAD: Anja Katrin Bielinsky

PROVIDER: PXD050381 | Pride | 2025-05-06

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	bieli003_chan1285_20161117_15691_drINC_A.mgf	Mgf
	bieli003_chan1285_20161117_15691_drINC_A.raw	Raw
	bieli003_chan1285_20161117_15691_drINC_B.mgf	Mgf
	bieli003_chan1285_20161117_15691_drINC_B.raw	Raw
	bieli003_chan1285_20161117_15691_drINC_K.mgf	Mgf

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Publications

Improved detection of DNA replication fork-associated proteins.

Rivard Rebecca S RS Chang Ya-Chu YC Ragland Ryan L RL Thu Yee-Mon YM Kassab Muzaffer M Mandal Rahul Shubhra RS Van Riper Susan K SK Kulej Katarzyna K Higgins LeeAnn L Markowski Todd M TM Shang David D Hedberg Jack J Erber Luke L Garcia Benjamin B Chen Yue Y Bielinsky Anja-Katrin AK Brown Eric J EJ

Cell reports 20240502 5

Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is ...[more]

PMID: 38703364

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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.