Differential DNA binding of Ku antigen determines its involvement in DNA replication.
Ku antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.
Schild-Poulter
Caroline
C
Department of Medicine, The Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada.
Matheos
Diamanto
D
Novac
Olivia
O
Cui
Bo
B
Giffin
Ward
W
Ruiz
Marcia T
MT
Price
Gerald B
GB
Zannis-Hadjopoulos
Maria
M
Haché
Robert J G
RJ
D034961:Antigens, Nuclear / Q000378:metabolism* / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics; D018983:DNA Footprinting; D004265:DNA Helicases; D004261:DNA Replication; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D018741:Replication Origin; D020218:Response Elements; D014466:Ultraviolet Rays
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen