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Comprehensive Interrogation of Synthetic Relationships in the Human DNA Damage Response

ABSTRACT: The DNA damage response (DDR) is a multi-faceted network of pathways that preserves genome stability. Unraveling the complementary interplay between these pathways remains a challenge. Here, we comprehensively mapped genetic interactions for all core DDR genes using combinatorial CRISPRi screening. We discovered myriad new connections, including interactions between cancer genes and small molecule targets. We focused on two of the strongest interactions: FEN1/LIG1:WDR48 and FANCM:SMARCAL1. First, we found that WDR48 works with USP1 to restrain overactive translesion synthesis in FEN1/LIG1-deficient cells, and that a preclinical inhibitor of USP1 specifically kills FEN1-deficient cells. Second, we found that SMARCAL1 and FANCM suppress DNA double-strand break (DSB) formation at TA-rich repeats in late-replicating regions that otherwise escape into mitosis and cause nuclear fragmentation. Our dataset provides a springboard for further mechanistic investigations into connections between DDR factors and suggests multiple interactions that could be exploited in cancer therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE236062 | GEO | 2025/02/03

REPOSITORIES: GEO

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Project description:Helicobacter pylori infection promotes chronic inflammation and plethora of DNA damages including strand breaks, base alterations, point mutations, microsatellite instability and epigenetic alterations. The gastric epitheliaI cells, in order to prevent genomic instability, require an integrous DNA damage repair (DDR) machinery which, however, has been reported to be modulated by the infection. CagA is a major Hp virulence factor that deregulates host cell functions such as proliferation, apoptosis and chromosomal integrity. Its pathogenic activity is partly regulated by tyrosine phosphorylation on repeated EPIYA-motifs. Our aim was to identify putative effects of H. pylori infection and CagA protein on DDR machinery, investigating the transcriptome of AGS cells, infected with P12 H. pylori wild-type strain, as well as, its corresponding CagA knock-out and the CagA phosphorylation-defective mutant following tyrosine (EPIYA) substitution by phenylalanine (EPIFA) in the EPIYA-C motifs. Upon RNA-Sequencing on polyA-selected transcripts we performed Differential Expression Analysis, Pathway Enrichment Analysis and visualization on KEGG Pathway Maps per DDR mechanism. Key DDR components that were observed to be downregulated in a CagA-related manner were validated via Western Blot utilizing AGS and the non-cancerous GES1 cell lines. Transcriptome analysis revealed that a notable number of DDR genes were downregulated during H. pylori infection resulting to potential modulation of Base Excision Repair, Mismatch Repair and a more intricate deregulation of Nucleotide Excision Repair, Homologous Recombination and Non-Homologous End-Joining. CagA contributes to MUTYH, FEN1, APE1, POLD1 and LIG1 downregulation and those observations were verified on the protein expression level, with the exception of APE1 that was on contrary observed to be upregulated. Our study accentuates the role of CagA, as a significant contributor of the H. pylori infection-mediated DDR modulation, disrupting the balance between DNA damage introduction and repair thus favoring genomic instability and potentially contributing to gastric carcinogenesis.

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Comprehensive Interrogation of Synthetic Relationships in the Human DNA Damage Response

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