Site-directed mutants of human RECQ1 reveal functional importance of the zinc binding domain

RecQ helicases are a highly conserved family of ATP-dependent DNA-unwinding enzymes with key roles in DNA replication and repair in all kingdoms of life. The RECQ1 gene encodes the most abundant RecQ homolog in humans. We engineered full-length RECQI harboring point mutations in the zinc-binding motif (amino acids 419-480) within the conserved RecQ-specific-C-terminal (RQC) domain known to be critical for diverse biochemical and cellular functions of RecQ helicases. Wild-type RECQI contains a zinc ion. Substitution of three of the four conserved cysteine residues that coordinate zinc severely impaired the ATPase and DNA unwinding activities but retained DNA binding and single strand DNA annealing activities. Furthermore, alteration of these residues attenuated zinc binding and significantly changed the overall conformation of full-length RECQ1 protein. In contrast, substitution of cysteine residue at position 471 resulted in a wild-type like RECQI protein. Differential contribution of the conserved cysteine residues to the structure and functions of the RECQI protein is also inferred by homology modeling. Overall, our results indicate that the zinc binding motif in the RQC domain of RECQ1 is a key structural element that is essential for the structure-functions of RECQI. Given the recent association of RECQI mutations with breast cancer, these results will contribute to understanding the molecular basis of RECQI functions in cancer etiology. (C) 2016 Elsevier B.V. All rights reserved.