DNA looping by Ku and the DNA-dependent protein kinase

Robert B. Cary, Scott R. Peterson, Jinting Wang, David G. Bear, E. Morton Bradbury, David J. Chen

Research output: Contribution to journalArticle

207 Scopus citations

Abstract

The DNA-dependent protein kinase (DNA-PK) is required for DNA double- strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70/86 heterodimer and an ≃460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and protein-nucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku dries not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self- association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.

Original languageEnglish (US)
Pages (from-to)4267-4272
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number9
DOIs
StatePublished - Apr 29 1997

Keywords

  • DNA repair
  • atomic force microscopy
  • double-strand break
  • electron microscopy

ASJC Scopus subject areas

  • General

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    Cary, R. B., Peterson, S. R., Wang, J., Bear, D. G., Bradbury, E. M., & Chen, D. J. (1997). DNA looping by Ku and the DNA-dependent protein kinase. Proceedings of the National Academy of Sciences of the United States of America, 94(9), 4267-4272. https://doi.org/10.1073/pnas.94.9.4267