Binding of the DNA-dependent protein kinase catalytic subunit to Holliday junctions.
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-PK(CS) (DNA-PK catalytic subunit). A comparison between different substrate architectures showed that DNA-PK(CS) 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-PK(CS) 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-PK(CS) 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-PK(CS) 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-PK(CS) 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.
Dip
Ramiro
R
Institute of Pharmacology and Toxicology, University of Zürich-Tierspital, Winterthurerstrasse 260, 8057 Zürich, Switzerland.
Naegeli
Hanspeter
H
D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain / Q000528:radiation effects; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry / Q000473:pathology; D003432:Cross-Linking Reagents / Q000378:metabolism; D004273:DNA, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D006801:Humans; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation / Q000528:radiation effects; D009698:Nucleoproteins / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics* / Q000528:radiation effects; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D001386:Azides; D002457:Cell Extracts; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009698:Nucleoproteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases