title: RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex.
abstract: RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a GFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to γ-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.
authors.affiliation: Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, NIH, 251 Bayview Boulevard, Baltimore, MD 21224, USA, INRA, Université de Toulouse, UMR1331, Toxalim, Research Centre in Food Toxicology, F-31027 Toulouse, France and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Raghavendra A
authors.initials: RA
authors.lastname: Shamanna
authors.affiliation: Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, NIH, 251 Bayview Boulevard, Baltimore, MD 21224, USA, INRA, Université de Toulouse, UMR1331, Toxalim, Research Centre in Food Toxicology, F-31027 Toulouse, France and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Dharmendra Kumar
authors.initials: DK
authors.lastname: Singh
authors.affiliation: Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, NIH, 251 Bayview Boulevard, Baltimore, MD 21224, USA, INRA, Université de Toulouse, UMR1331, Toxalim, Research Centre in Food Toxicology, F-31027 Toulouse, France and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Huiming
authors.initials: H
authors.lastname: Lu
authors.affiliation: INRA, Université de Toulouse, UMR1331, Toxalim, Research Centre in Food Toxicology, F-31027 Toulouse, France and.
authors.forename: Gladys
authors.initials: G
authors.lastname: Mirey
authors.affiliation: Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Guido
authors.initials: G
authors.lastname: Keijzers
authors.affiliation: INRA, Université de Toulouse, UMR1331, Toxalim, Research Centre in Food Toxicology, F-31027 Toulouse, France and.
authors.forename: Bernard
authors.initials: B
authors.lastname: Salles
authors.affiliation: Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, NIH, 251 Bayview Boulevard, Baltimore, MD 21224, USA, INRA, Université de Toulouse, UMR1331, Toxalim, Research Centre in Food Toxicology, F-31027 Toulouse, France and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Croteau
authors.affiliation: Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, NIH, 251 Bayview Boulevard, Baltimore, MD 21224, USA, INRA, Université de Toulouse, UMR1331, Toxalim, Research Centre in Food Toxicology, F-31027 Toulouse, France and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark vbohr@nih.gov.
authors.forename: Vilhelm A
authors.initials: VA
authors.lastname: Bohr
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D059766:DNA End-Joining Repair / Q000235:genetics*; D051747:DNA-Activated Protein Kinase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005720:Gamma Rays; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D000072200:Ku Autoantigen; D011836:Radiation Tolerance / Q000235:genetics; D053484:RecQ Helicases / Q000037:antagonists & inhibitors / Q000235:genetics*; D011038:Rothmund-Thomson Syndrome / Q000235:genetics* / Q000473:pathology; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D051747:DNA-Activated Protein Kinase; C506731:RECQL4 protein, human; D053484:RecQ Helicases; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen