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D-type cyclins determine the activity of DNA mismatch repair in the G1 and S phases of the cell cycle

ABSTRACT: The large majority of oxidative lesions occurring in the G1 phase of the cell cycle are repaired by base excision repair (BER) rather than mismatch repair (MMR) to avoid long resections that can lead to genomic instability and cell death. However, how cells choose BER over MMR is not yet understood. Here, we show that, during G1, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. In turn, in a manner that is independent on CDK4/6 activity, D-type cyclins stabilize p21, which competes through its PCNA-interacting protein (PIP) box with MMR components for their binding to PCNA. This reduces MMR activity while allowing BER. At the G1/S transition, the AMBRA1-dependent degradation of D-type cyclins renders p21 susceptible to proteolysis via SKP2 and CDT2. These timely degradation events allow the proper binding of MMR proteins to PCNA enabling the repair of DNA replication errors. Thus, the expression of D-type cyclins limit MMR in G1, whereas their degradation is necessary for proper MMR function in S. Defects in these two regulatory mechanisms promote genome instability. The mass spectrometry raw files correspond to the affinity purifications of proximity labeled (turbo-ID) PCNA and CCND1 under various conditions.

INSTRUMENT(S): Q Exactive HF-X, Q Exactive

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

SUBMITTER: Beatrix Ueberheide

PROVIDER: MSV000091006 | MassIVE | Tue Jan 03 14:48:00 GMT 2023

SECONDARY ACCESSION(S): PXD039228

REPOSITORIES: MassIVE

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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.

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D-type cyclins determine the activity of DNA mismatch repair in the G1 and S phases of the cell cycle

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