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. Mutations in RECQ1 significantly increase breast cancer susceptibility. 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 observations will contribute to understanding the molecular basis of RECQ1 functions in cancer etiology.

The Human RecQ4 Helicase Contains a Functional RecQ C-terminal Region (RQC) That Is Essential for Activity

“…When two of the Escherichia coli RecQ zinc ligands are mutated to Asn, the DNA binding and helicase activity is abolished, whereas ATPase activity is retained (25); similarly, mutations of the zinc ligands in BLM result in either insoluble or biochemically compromised protein, where DNA binding, ATPase, and helicase activities are compromised (26). A more recent paper suggests that mutations in three of the cysteine residues that coordinate zinc affect ATP binding, DNA binding, ATPase, and helicase activity but not strand annealing (27); furthermore the same three cysteine mutants exhibit enhanced thermal denaturation. Our results overlap only in part with previous work, showing that the cysteine mutants in RecQ4 have the same thermal stability as the wild type, do not impair the ATPase activity (in excess of stimulatory DNA), but affect nucleic acid binding, annealing, and helicase activity (Fig.…”

RECQ1 Helicase in Genomic Stability and Cancer

“…RECQ1 protein forms two quaternary structures: the higher-order oligomers consistent with hexamers and pentamers are associated with single-strand DNA annealing, whereas lower-order oligomers, consistent with dimers and monomers, are associated with duplex DNA unwinding [ 13 ]. The observation that the truncated RECQ1 (amino acid residues 49–616) lacks a single-strand annealing property indicates that the oligomerization signals for RECQ1 are in the N-terminal region [ 14 , 15 , 16 ].…”

“…The ZBD consists of highly conserved 4 cysteine zinc-binding motif and two anti-parallel α-helices (HH) [ 14 ] ( Figure 1 A). Alteration of these cysteine residues changes the overall conformation of RECQ1 protein and impairs its helicase and ATPase activities, whereas the strand-annealing activity is minimally affected [ 15 , 16 ]. The WH domain of RECQ1 has a β-hairpin loop and a tyrosine residue (Y564) at the tip of the hairpin, which is important for the DNA unwinding and ATPase activity, indicating the importance of this region and the residue in the enzyme’s helicase function [ 14 ].…”

The DNA repair helicase RECQ1 has a checkpoint-dependent role in mediating DNA damage responses induced by gemcitabine

“…To compare the functions of the WT RECQ1 protein with those of the RECQ1 variant in an isogenic background, we reconstituted the RECQ1-KO cell line using engineered pCB6 vectors that stably expressed either the empty vector or the full-length WT RECQ1 or RECQ1 variants with individual missense mutations (A195S, R215Q, R455C, M458K, and T562I), which were reported to increase breast cancer susceptibility. Based on the crystal structure and conserved functions of RecQ helicases (1), Ala-195 is involved in dimer interaction (31); Arg-215 is located near the ADP-binding pocket and is expected to weaken ATP hydrolysis (32,33); the conserved residues Arg-455 and Met-458 are located in the zinc-binding subdomain, which is important in maintaining protein stability (34); and Thr-562 is located in a ␤-hairpin, which is required for DNA unwinding (31,35). Compared with the ability of WT RECQ1 helicase to unwind forked DNA substrates, the RECQ1 variants R215Q, R455C, M458K, and T562I show complete loss of helicase activity, and A195S variant shows very weak helicase activity in vitro (6).…”

“…Compared with the ability of WT RECQ1 helicase to unwind forked DNA substrates, the RECQ1 variants R215Q, R455C, M458K, and T562I show complete loss of helicase activity, and A195S variant shows very weak helicase activity in vitro (6). Therefore, we also included a K119R variant that has not yet been associated with cancer risk but has been biochemically characterized as helicase-dead (9,32).…”