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

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 delta 4 (Pol delta 4) 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 delta 4 and/or PCNA. DNA synthesis by Pol delta 4 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 delta 4 was monitored by incorporation of [alpha-P-32]dNTPs into the 93mer primer followed by denaturing gel electrophoresis. The results showed that DHX9 strongly stimulated Pol delta 4 mediated D-loop extension. Direct interactions of DHX9 with PCNA, the p125 and the p12 subunits of Pol delta 4 were demonstrated by pull-down assays with purified proteins. These data support the hypothesis that DHX9 helicase is recruited by Pol delta 4/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.

Automated summary

The study aimed to test the hypotheses that D-loop extension in homology directed repair (HDR) is facilitated by DHX9 and that its recruitment is mediated by protein-protein interactions between DHX9 and Pol delta 4 and/or PCNA. The results showed that DHX9 strongly stimulated Pol delta 4 mediated D-loop extension and direct interactions between DHX9 and PCNA as well as Pol delta 4 subunits were observed. These findings suggest that DHX9 is recruited by Pol delta 4/PCNA to facilitate D-loop synthesis in HDR and plays a role in cellular HDR.