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Michael Wallisch, Elisabeth Kunkel, Karsten Hoehn, Friedrich Grummt
Ku Antigen Supports Termination of Mammalian rDNA Replication by Transcription Termination Factor TTF-I
A replication fork barrier at the 3end 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 DNAprotein complex including transcription termination factor I, and specific proteinprotein interactions occur between this factor and protein(s) of the replication machinery. Here we report that a second DNAbinding 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 GCstretch 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 DNAbinding heterodimer that is the target of autoimmuneantibodies in several autoimmune diseases. Recombinant Ku protein, purified as heterodimer from coinfected insect cells, is able to partially rescue the barrier activity in Kudepleted HeLa cell extracts. These data demonstrate that transcription termination factor I and Ku act synergistically to prevent headon collision between the replication and the transcription machinery.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 383, 05/2002
Pages: 765 - 771