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

ABSTRACT: RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency, and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a RecQ C-terminal region (RQC) found in the other RecQ paralogues; such a region consists of a zinc domain and a winged helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. Inductively coupled plasma-atomic emission spectrometry detected the unusual presence of two zinc clusters within the zinc domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative zinc ligands and the aromatic residue predicted to be at the tip of the winged helix ?-hairpin), showed a decrease in DNA binding, unwinding, and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.

SUBMITTER: Mojumdar A

PROVIDER: S-EPMC5354486 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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

Mojumdar Aditya A De March Matteo M Marino Francesca F Onesti Silvia S

The Journal of biological chemistry 20161220 10

RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency, and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a RecQ C ...[more]

PMID: 27998982

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Project description:BACKGROUND:Pyrococcus furiosus Hjm (PfuHjm) is a structure-specific DNA helicase that was originally identified by in vitro screening for Holliday junction migration activity. It belongs to helicase superfamily 2, and shares homology with the human DNA polymerase Theta (PolTheta), HEL308, and Drosophila Mus308 proteins, which are involved in DNA repair. Previous biochemical and genetic analyses revealed that PfuHjm preferentially binds to fork-related Y-structured DNAs and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial RecQ helicase, which is essential for genome maintenance. Elucidation of the DNA unwinding and translocation mechanisms by PfuHjm will require its three-dimensional structure at atomic resolution. RESULTS:We determined the crystal structures of PfuHjm, in two apo-states and two nucleotide bound forms, at resolutions of 2.0-2.7 A. The overall structures and the local conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, irrespective of the nucleotide-binding states. The architecture of Hjm was similar to that of Archaeoglobus fulgidus Hel308 complexed with DNA. An Hjm-DNA complex model, constructed by fitting the five domains of Hjm onto the corresponding Hel308 domains, indicated that the interaction of Hjm with DNA is similar to that of Hel308. Notably, sulphate ions bound to Hjm lie on the putative DNA binding surfaces. Electron microscopic analysis of an Hjm-DNA complex revealed substantial flexibility of the double stranded region of DNA, presumably due to particularly weak protein-DNA interactions. Our present structures allowed reasonable homology model building of the helicase region of human PolTheta, indicating the strong conformational conservation between archaea and eukarya. CONCLUSION:The detailed comparison between our DNA-free PfuHjm structure and the structure of Hel308 complexed with DNA suggests similar DNA unwinding and translocation mechanisms, which could be generalized to all of the members in the same family. Structural comparison also implied a minor rearrangement of the five domains during DNA unwinding reaction. The unexpected small contact between the DNA duplex region and the enzyme appears to be advantageous for processive helicase activity.

Dataset Information

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

Publications

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

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