The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells

Abstract

SUMMARY Replication-blocking DNA lesions are particularly toxic to proliferating cells because they can lead to chromosome mis-segregation if not repaired prior to mitosis. In this study, we report that ZGRF1 null cells accumulate chromosome aberrations following replication perturbation and show sensitivity to two potent replication-blocking anticancer drugs: mitomycin C and camptothecin. Moreover, ZGRF1 null cells are defective in catalyzing DNA damage-induced sister chromatid exchange despite accumulating excessive FANCD2, RAD51, and γ-H2AX foci upon induction of interstrand DNA crosslinks. Consistent with a direct role in promoting recombinational DNA repair, we show that ZGRF1 is a 5′-to-3′ helicase that catalyzes D-loop dissociation and Holliday junction branch migration. Moreover, ZGRF1 physically interacts with RAD51 and stimulates strand exchange catalyzed by RAD51-RAD54. On the basis of these data, we propose that ZGRF1 promotes repair of replication-blocking DNA lesions through stimulation of homologous recombination.

Protocol for Purification of Human ZGRF1 and Its Regulatory Function on RAD51-Mediated D-Loop Formation

“…The D-loop formation assay provides a powerful tool for investigating the presynaptic HR role of human ZGRF1. With the optimized protocol, we are able to clearly demonstrate that ZGRF1 enhances the RAD51-mediated D-loop formation ( Figure 3 and Brannvoll et al, 2020 ). This biochemical result is consistent with our finding that knockout of ZGRF1 by CRISPR-Cas9 leads to a lower HR efficiency in cells as measured using the DR-GFP reporter ( Brannvoll et al, 2020 ).…”

“…ZGRF1 knockdown or knockout impairs HR efficiency, indicating its role therein ( Adamson et al, 2012 , Brannvoll et al, 2020 ). A D-loop formed between ssDNA and a donor duplex DNA is a key intermediate in the repair of DNA double-strand breaks or damaged DNA replication forks by HR.…”

“…Human ZGRF1 (zinc finger GRF-type containing 1, C4orf21) gene encodes a putative DNA/RNA helicase that promotes the removal of replication-blocking DNA lesions ( Brannvoll et al, 2020 ). ZGRF1 protein is composed of 2,104 amino acid residues, and it contains a DUF2439 domain, a (GRF-type) Zn-finger domain, and a helicase domain.…”

“…We have also tuned biochemical assays by lowering the protein and DNA concentrations to analyze its functions in RAD51-mediated D-loop formation and dissociation. For complete details on the use and execution of this protocol, please refer to Brannvoll et al (2020) .…”

“…For complete details on the use and execution of this protocol, please refer to Brannvoll et al (2020) .…”