Dataset Information

FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress

ABSTRACT: Oncogene-induced replication stress constitutes an early obstacle for pre-cancerous cells to overcome to progress towards malignancy. Fanconi anaemia signalling represents a major genomic maintenance pathway that is activated in response to replication stress, impinging on stalled replication fork stability and recovery. We report that upon replication stress, phosphorylation of the FANCD2 N-terminus by CHK1 triggers FBXL12-dependent proteasomal degradation of FANCD2, facilitating clearance of FANCD2 at stalled replication forks and promoting replication recovery. Monoubiquitination of FANCD2 at K561 by the Fanconi anaemia core complex regulates clamping of the FANCD2-FANCI heterodimer onto DNA. To investigate if K561-monoubiquitination influences FANCD2 phosphorylation and/or polyubiquitination by FBXL12, we performed MS-based proteomics using HEK293 cells co-transfected with the monoubiquitin-deficient FANCD2 mutant (K561R) and FANCI, or a CHK1 phosphorylation-deficient FANCD2-AA (S8A/S10A) mutant as control. To induce rapid activation of CHK1, we inhibited WEE1 kinase using AZD1775, immunoprecipitated FANCD2 from chromatin extracts and performed MS analysis. The MS data revealed several phosphorylated N-terminal peptides in the K561R-FANCD2 sample, including S8, S10 and also S15 demonstrating that K561R is phosphorylated in response to replication stress induced by AZD1775.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Georgios Mermelekas

LAB HEAD: Janne Lehtiö

PROVIDER: PXD043637 | Pride | 2025-05-06

REPOSITORIES: Pride

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			Action	DRS
	221028_GMLO-Andra_IPMS_5of15ul_3hrrgad_sample01.raw	Raw
	221028_GMLO-Andra_IPMS_5of15ul_3hrrgad_sample02.raw	Raw
	221028_GMLO-Andra_IPMS_5of15ul_3hrrgad_sample05.raw	Raw
	221101_GMLO-Andra_IPMS_protein_results.xlsx	Xlsx
	checksum.txt	Txt

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FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress.

Brunner Andrä A Li Qiuzhen Q Fisicaro Samuele S Kourtesakis Alexandros A Viiliäinen Johanna J Johansson Henrik J HJ Pandey Vijaya V Mayank Adarsh K AK Lehtiö Janne J Wohlschlegel James A JA Spruck Charles C Rantala Juha K JK Orre Lukas M LM Sangfelt Olle O

Molecular cell 20230816 20

Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to ...[more]

PMID: 37591242

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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.