RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex

Raghavendra A. Shamanna, Dharmendra K.umar Singh, Huiming Lu, Gladys Mirey, Guido Keijzers, Bernard Salles, Deborah L. Croteau, Vilhelm A. Bohr

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2415-2424
Number of pages10
JournalCarcinogenesis
Volume35
Issue number11
DOIs
StatePublished - Nov 1 2014
Externally publishedYes

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RecQ Helicases
DNA-Activated Protein Kinase
DNA
Inborn Genetic Diseases
Double-Stranded DNA Breaks
Cytoplasm
Lasers
Plasmids
Mutation
Proteins
In Vitro Techniques

ASJC Scopus subject areas

  • Cancer Research

Cite this

Shamanna, R. A., Singh, D. K. U., Lu, H., Mirey, G., Keijzers, G., Salles, B., ... Bohr, V. A. (2014). RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex. Carcinogenesis, 35(11), 2415-2424. https://doi.org/10.1093/carcin/bgu137

RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex. / Shamanna, Raghavendra A.; Singh, Dharmendra K.umar; Lu, Huiming; Mirey, Gladys; Keijzers, Guido; Salles, Bernard; Croteau, Deborah L.; Bohr, Vilhelm A.

In: Carcinogenesis, Vol. 35, No. 11, 01.11.2014, p. 2415-2424.

Research output: Contribution to journalArticle

Shamanna, RA, Singh, DKU, Lu, H, Mirey, G, Keijzers, G, Salles, B, Croteau, DL & Bohr, VA 2014, 'RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex', Carcinogenesis, vol. 35, no. 11, pp. 2415-2424. https://doi.org/10.1093/carcin/bgu137
Shamanna, Raghavendra A. ; Singh, Dharmendra K.umar ; Lu, Huiming ; Mirey, Gladys ; Keijzers, Guido ; Salles, Bernard ; Croteau, Deborah L. ; Bohr, Vilhelm A. / RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex. In: Carcinogenesis. 2014 ; Vol. 35, No. 11. pp. 2415-2424.
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AU - Singh, Dharmendra K.umar

AU - Lu, Huiming

AU - Mirey, Gladys

AU - Keijzers, Guido

AU - Salles, Bernard

AU - Croteau, Deborah L.

AU - Bohr, Vilhelm A.

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N2 - 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.

AB - 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.

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