Mechanism of interaction between Ku protein and DNA

T. Mimori, J. A. Hardin

Research output: Contribution to journalArticle

355 Citations (Scopus)

Abstract

The mechanism of interaction between the Ku autoantigenic protein, a heterodimer of noncovalently linked 70,000- and 80,000-dalton subunits, and DNA was studied using immunoaffinity-purified Ku protein and a 300-base pair EcoRI fragment from HeLa cell DNA. In the nitrocellulose filter-binding assay, the Ku protein bound 32P-labeled double-stranded DNA, and much less efficiently single-stranded DNA. The binding of Ku to DNA was dependent on ionic strength and prevented by IgG from patient sera containing anti-Ku antibodies. In competitive assays, using unlabeled nucleic acid competitors, the DNA binding of Ku was not inhibited in the presence of yeast tRNA, synthetic copolymer of poly(A)-poly(dT), or circular plasmid pBR322 DNA, but was inhibited when the plasmid DNA was cleaved with appropriate restriction endonucleases. The inhibitory activities of cleaved plasmid DNA were independent of the configuration or nucleotide sequences at ends but proportional to the number of recognition sites of restriction enzymes used. Footprint analysis demonstrated that Ku protein protected both 3'- and 5'-terminal regions of double-stranded DNA from DNase I digestion. When Ku protein was fractionated electrophoretically, transferred to nitrocellulose filter, and probed with 32P-labeled DNA, only the 70,000-dalton subunit exhibited DNA binding. Thus, the Ku protein appears to recognize selectively ends of double-stranded DNA molecules. Possible functions of the Ku autoantigen in eukaryotic cells are discussed.

Original languageEnglish (US)
Pages (from-to)10375-10379
Number of pages5
JournalJournal of Biological Chemistry
Volume261
Issue number22
StatePublished - 1986
Externally publishedYes

Fingerprint

DNA
Proteins
Plasmids
Collodion
Ku Autoantigen
Assays
Poly T
Poly A
Single-Stranded DNA
Deoxyribonuclease I
DNA Restriction Enzymes
Autoantigens
Eukaryotic Cells
Transfer RNA
Ionic strength
HeLa Cells
Base Pairing
Yeast
Osmolar Concentration
Nucleic Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mimori, T., & Hardin, J. A. (1986). Mechanism of interaction between Ku protein and DNA. Journal of Biological Chemistry, 261(22), 10375-10379.

Mechanism of interaction between Ku protein and DNA. / Mimori, T.; Hardin, J. A.

In: Journal of Biological Chemistry, Vol. 261, No. 22, 1986, p. 10375-10379.

Research output: Contribution to journalArticle

Mimori, T & Hardin, JA 1986, 'Mechanism of interaction between Ku protein and DNA', Journal of Biological Chemistry, vol. 261, no. 22, pp. 10375-10379.
Mimori, T. ; Hardin, J. A. / Mechanism of interaction between Ku protein and DNA. In: Journal of Biological Chemistry. 1986 ; Vol. 261, No. 22. pp. 10375-10379.
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