The DHX9 helicase interacts with human DNA polymerase δ4 and stimulates its activity in D-loop extension synthesis

• Published in: DNA repair 2023-08, Vol.128, p.103513-103513, Article 103513
• Main Authors: Wang, Xiaoxiao, Zhang, Sufang, Zhang, Zhongtao, Mazloum, Nayef A., Lee, Ernest Y.C., Lee, Marietta Y.W.
• Format: Article
• Language: English
• Subjects: D-loop synthesis; DEAD-box RNA Helicases - metabolism; DHX9 helicase; DNA double-strand break repair; DNA Helicases - metabolism; DNA Polymerase III - genetics; DNA polymerase δ; DNA Replication; DNA-Directed DNA Polymerase - metabolism; Helicase-polymerase interaction; Homologous recombination; Humans; Neoplasm Proteins - genetics; Proliferating Cell Nuclear Antigen - metabolism
• ISSN: 1568-7864; 1568-7856; 1568-7856
• DOI: 10.1016/j.dnarep.2023.103513

The extension of the invading strand within a displacement loop (D-loop) is a key step in homology directed repair (HDR) of doubled stranded DNA breaks. The primary goal of these studies was to test the hypotheses that 1) D-loop extension by human DNA polymerase δ4 (Pol δ4) is facilitated by DHX9, a 3′ to 5′ motor helicase, which acts to unwind the leading edge of the D-loop, and 2) the recruitment of DHX9 is mediated by direct protein-protein interactions between DHX9 and Pol δ4 and/or PCNA. DNA synthesis by Pol δ4 was analyzed in a reconstitution assay by the extension of a 93mer oligonucleotide inserted into a plasmid to form a D-loop. Product formation by Pol δ4 was monitored by incorporation of [α-32P]dNTPs into the 93mer primer followed by denaturing gel electrophoresis. The results showed that DHX9 strongly stimulated Pol δ4 mediated D-loop extension. Direct interactions of DHX9 with PCNA, the p125 and the p12 subunits of Pol δ4 were demonstrated by pull-down assays with purified proteins. These data support the hypothesis that DHX9 helicase is recruited by Pol δ4/PCNA to facilitate D-loop synthesis in HDR, and is a participant in cellular HDR. The involvement of DHX9 in HDR represents an important addition to its multiple cellular roles. Such helicase-polymerase interactions may represent an important aspect of the mechanisms involved in D-loop primer extension synthesis in HDR. [Display omitted] •DHX9 helicase facilitates D-loop extension synthesis in double-strand break repair.•DHX9 stimulates DNA synthesis by Pol δ4 in the D-loop.•DHX9 exhibits direct protein-protein interactions with Pol δ4 and PCNA.•DHX9 is mechanistically involved in double-strand break repair.•This new role of DHX9 adds to its potential as a target for cancer therapeutics.