The removal of DNA interstrand crosslinks (ICLs) is vital for maintenance of genome integrity. ICL repair is aided by the Fanconi anemia-associated FANCM helicase. In Saccharomyces cerevisiae the FANCM orthologue is Mph1. Our lab has identified Mte1 (Mph1-associated telomere maintenance protein 1) as a novel interactor of Mph1 in budding yeast. Mte1 stimulates the fork regression and helicase activities of Mph1 while inhibiting Mph1-catalyzed D-loop dissociation. Deletion of MTE1 results in reduced crossover recombination and supresses the replication stress sensitivity of mph1∆ mutant cells. Mte1-Mph1 is recruited preferentially to telomeres and mte1∆ mutants suffer over-extension of telomeres, suggesting that DNA replication stress at telomeres is a substrate for Mte1-Mph1.
ZGRF1 (Zinc Finger GRF-Type Containing 1) is the human orthologue of the Mte1-Mph1 complex. To investigate the roles of ZGRF1, we knocked out ZGRF1 in the HCT116, USO2 and RPE-1 human cell lines using CRISPR-Cas9 genome editing system. ZGRF1-deleted cells exhibit increased sensitivity to the DNA crosslinking agent Mitomycin C (MMC) and the Topoisomerase I inhibitor Camptothecin (CPT), but not to ionizing or UV-irradiation. Moreover, treatment with MMC results in increased chromosomal aberrations in the ZGRF1 null cell line, and decreased sister-chromatid exchange is observed following treatment with MMC or CPT. In response to MMC, the ZGRF1-deleted cells exhibit an increase in co-localization of the FA protein FANCD2 and the DNA double-strand break marker gamma-H2AX. In vitro, purified ZGRF1 is a 5’ to 3’ helicase that catalyzes ATP-dependent R- and D-loop displacement, Holliday junction branch migration and fork regression.
Tags: Biology, Genetics, DNA replication, Molecular genetics, Senescence, Helicase, DNA, RecQ helicase, Werner syndrome
Annonce publiée le 18-10-2018
Centre de Recherche - Paris - Amphitheatre Antoine Lacassagne