Our DNA is constantly damaged by external chemical or radiation attack, or by intracellular by-products of metabolism. As such, cells have evolved complex mechanisms of DNA damage repair. Insufficient DNA repair and the accumulation of mutations drives aging and diseases such as cancer. FANCM is an integral DNA repair protein that works specifically at damage that blocks DNA replication. The major function of FANCM is as a DNA translocase that binds and remodels branched DNA structures such as a stalled replication fork with high affinity. Through several protein:protein interactions, FANCM also recruits downstream DNA repair factors. These roles in DNA repair are closely linked to FANCM’s function as a tumour suppressor protein in several different cancer settings.
We used recombinant FANCM protein and in vitro biochemical assays to show that the N-terminal translocase domain of FANCM is responsible for the recognition and ATP-dependent migration of branched DNA structures. An ‘insert’ domain within the translocase acts as the substrate-specificity domain responsible for FANCMs high affinity to branched substrates. We show that deletion of the insert domain disrupts the ability of FANCM to functionally engage its substrate. Three residues within the insert - K331, Y332, and H369 - were identified. Mutation of these residues perturbs ‘branch migration activity’.
Our results demonstrate how FANCM couples branched DNA binding to ATPase activity required to remodel critical structures in DNA replication and repair. These findings provide novel insight into the mechanism of cancer suppression by FANCM.