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BRCA1 suppresses microhomology-mediated tandem duplications at Tus/Ter-stalled replication forks

ABSTRACT: Small ~10 kb microhomology-mediated tandem duplications (“Group 1 TDs”) are abundant in BRCA1-linked but not BRCA2-linked breast cancer genomes. Here, we define the mechanism underlying this rearrangement signature. We show that BRCA1, but not BRCA2, suppresses TDs at a Tus/Ter site-specific chromosomal replication fork barrier in primary mammalian cells. BRCA1 has no equivalent role at chromosomal double strand breaks, indicating specificity for the stalled fork response. Two motor proteins—FANCM and the Bloom’s syndrome helicase—suppress Tus/Ter-induced TDs in BRCA1 mutants, revealing the existence of a multi-gene TD suppressor network. TDs arise by a “replication restart-bypass” mechanism terminated by end joining or microhomology-mediated template switching, the latter forming complex TD breakpoints. We show that solitary DNA ends form directly at Tus/Ter, implicating misrepair of these lesions in TD formation. We find that BRCA1 inactivation is strongly associated with Group 1 TDs in ovarian cancer. The Group 1 TD phenotype may be a general signature of BRCA1-deficient cancer

ORGANISM(S): Mus musculus

PROVIDER: GSE103624 | GEO | 2017/11/23

SECONDARY ACCESSION(S): PRJNA403795

REPOSITORIES: GEO

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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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BRCA1 suppresses microhomology-mediated tandem duplications at Tus/Ter-stalled replication forks

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