title: Site-directed mutants of human RECQ1 reveal functional importance of the zinc binding domain.
abstract: 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 RECQ1 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 RECQ1 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 RECQ1 protein. Differential contribution of the conserved cysteine residues to the structure and functions of the RECQ1 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 RECQ1. Given the recent association of RECQ1 mutations with breast cancer, these results will contribute to understanding the molecular basis of RECQ1 functions in cancer etiology.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520 W Street NW, Washington DC 20059, USA.
authors.forename: Furqan
authors.initials: F
authors.lastname: Sami
authors.affiliation: Department of Chemistry and Biochemistry, University of Nevada, 4505 Maryland Parkway, Las Vegas, NV 89154-4003, USA.
authors.forename: Ronald K
authors.initials: RK
authors.lastname: Gary
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520 W Street NW, Washington DC 20059, USA.
authors.forename: Yayin
authors.initials: Y
authors.lastname: Fang
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520 W Street NW, Washington DC 20059, USA. Electronic address: sudha.sharma@howard.edu.
authors.forename: Sudha
authors.initials: S
authors.lastname: Sharma
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D000072417:Protein Domains / Q000235:genetics*; D053484:RecQ Helicases / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C017800:zinc-binding protein; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C506725:RECQL protein, human; D053484:RecQ Helicases
keywords: Helicase; Mutation; RecQ; Strand-annealing; Zinc-binding domain