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Binding of the DNA-dependent protein kinase catalytic subunit to holliday junctions


Dip, R; Naegeli, H (2004). Binding of the DNA-dependent protein kinase catalytic subunit to holliday junctions. Biochemical Journal, 381(Pt. 1):165-174.

Abstract

DNA-PK (DNA-dependent protein kinase) is a double-strand break sensor involved in DNA repair and signal transduction. In the present study, we constructed site-directed cross-linking probes to explore the range of DNA discontinuities that are recognized by DNA-PKCS (DNA-PK catalytic subunit). A comparison between different substrate architectures showed that DNA-PKCS associates preferentially with the crossover region of synthetic Holliday junctions. This interaction with four-way junctions was preserved when biotin–streptavidin complexes were assembled at the termini to exclude the binding of Ku proteins. The association of DNA-PKCS with Holliday junctions was salt-labile even in the presence of Ku proteins, but this interaction could be stabilized when the DNA probes were incubated with the endogenous enzyme in nuclear extracts of human cells. Cross-linking of the endogenous enzyme in cellular extracts also demonstrated that DNA-PKCS binds to DNA ends and four-way junctions with similar affinities in the context of a nuclear protein environment. Kinase assays using p53 proteins as a substrate showed that, in association with four-way structures, DNA-PKCS adopts an active conformation different from that in the complex with linear DNA. Our results are consistent with a structure-specific, but Ku- and DNA end-independent, recruitment of DNA-PKCS to Holliday junction intermediates. This observation suggests an unexpected functional link between the two main pathways that are responsible for the repair of DNA double-strand breaks in mammalian cells.

Abstract

DNA-PK (DNA-dependent protein kinase) is a double-strand break sensor involved in DNA repair and signal transduction. In the present study, we constructed site-directed cross-linking probes to explore the range of DNA discontinuities that are recognized by DNA-PKCS (DNA-PK catalytic subunit). A comparison between different substrate architectures showed that DNA-PKCS associates preferentially with the crossover region of synthetic Holliday junctions. This interaction with four-way junctions was preserved when biotin–streptavidin complexes were assembled at the termini to exclude the binding of Ku proteins. The association of DNA-PKCS with Holliday junctions was salt-labile even in the presence of Ku proteins, but this interaction could be stabilized when the DNA probes were incubated with the endogenous enzyme in nuclear extracts of human cells. Cross-linking of the endogenous enzyme in cellular extracts also demonstrated that DNA-PKCS binds to DNA ends and four-way junctions with similar affinities in the context of a nuclear protein environment. Kinase assays using p53 proteins as a substrate showed that, in association with four-way structures, DNA-PKCS adopts an active conformation different from that in the complex with linear DNA. Our results are consistent with a structure-specific, but Ku- and DNA end-independent, recruitment of DNA-PKCS to Holliday junction intermediates. This observation suggests an unexpected functional link between the two main pathways that are responsible for the repair of DNA double-strand breaks in mammalian cells.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Institute of Veterinary Pharmacology and Toxicology
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2004
Deposited On:27 Mar 2009 16:10
Last Modified:06 Dec 2017 18:06
Publisher:Biochemical Society
ISSN:0264-6021
Funders:Swiss National Science Foundation
Publisher DOI:https://doi.org/10.1042/BJ20031666
PubMed ID:15035658

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