Ku antigen supports termination of mammalian rDNA replication by transcription termination factor TTF-I.
A replication fork barrier at the 3'-end of mouse ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. This barrier is an inherent property of a defined DNA-protein complex including transcription termination factor I, and specific protein-protein interactions occur between this factor and protein(s) of the replication machinery. Here we report that a second DNA-binding protein is essential for barrier activity. We have purified and functionally characterised the protein from HeLa cells. The final preparation contained two polypeptides with molecular masses of 70 and 86 kDa, respectively. Both polypeptides interact with a GC-stretch adjacent to the binding site of transcription termination factor I. The specificity of binding to the barrier DNA was demonstrated in an electrophoretic mobility shift assay. The biochemical properties of this protein resemble that of Ku antigen, a human nuclear DNA-binding heterodimer that is the target of autoimmune-antibodies in several autoimmune diseases. Recombinant Ku protein, purified as heterodimer from co-infected insect cells, is able to partially rescue the barrier activity in Ku-depleted HeLa cell extracts. These data demonstrate that transcription termination factor I and Ku act synergistically to prevent head-on collision between the replication and the transcription machinery.
Wallisch
Michael
M
Institute of Biochemistry, University of Würzburg, Germany.
Kunkel
Elisabeth
E
Hoehn
Karsten
K
Grummt
Friedrich
F
D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004265:DNA Helicases; D004261:DNA Replication / Q000502:physiology*; D004275:DNA, Ribosomal / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007313:Insecta; D000072200:Ku Autoantigen; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000378:metabolism; D018741:Replication Origin / Q000502:physiology; D013379:Substrate Specificity; D014157:Transcription Factors
D034961:Antigens, Nuclear; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D021122:Protein Subunits; D011994:Recombinant Proteins; C504003:TTF1 protein, human; D014157:Transcription Factors; C504004:Ttf1 protein, mouse; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen