Human Ku autoantigen binds cisplatin-damaged DNA but fails to stimulate human DNA-activated protein kinase

John Turchi, Karen Henkels

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

We have identified a series of proteins based on an affinity for cisplatin-damaged DNA. One protein termed DRP-1 has been purified to homogeneity and was isolated as two distinct complexes. The first complex is a heterodimer of 83- and 68-kDa subunits, while the second complex is a heterotrimer of 350-, 83-, and 68-kDa subunits in a 1:1:1 ratio. The 83- and 68-kDa subunits in each complex are identical. The 83-kDa subunit of DRP-1 was identified as the p80 subunit of Ku autoantigen by N-terminal protein sequence analysis and reactivity with a monoclonal antibody directed against human Ku p80 subunit. The 68-kDa subunit of DRP-1 cross-reacted with monoclonal antisera raised against the Ku autoantigen p70 subunit. The 350- kDa subunit was identified as DNA-PK(cs), the catalytic subunit of the human DNA-activated protein kinase, DNA-PK. DRP-1/Ku DNA binding was assessed in mobility shift assays and competition binding assays using cisplatin-damaged DNA. Results indicate that DNA binding was essentially unaffected by cisplatin-DNA adducts in the presence or absence of DNA-PK(cs). DNA-PK activity was only stimulated with undamaged DNA, despite the ability of Ku to bind to cisplatin-damaged DNA. The lack of DNA-PK stimulation by cisplatin- damaged DNA correlated with the extent of cisplatin-DNA adduct formation. These results demonstrate that Ku can bind cisplatin-damaged DNA but fails to activate DNA-PK. These results are discussed with respect to the repair of cisplatin-DNA adducts and the role of DNA-PK in coordinating DNA repair processes.

Original languageEnglish (US)
Pages (from-to)13861-13867
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number23
DOIs
StatePublished - 1996
Externally publishedYes

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DNA-Activated Protein Kinase
Autoantigens
Cisplatin
DNA
Ku Autoantigen
Assays
Repair
Proteins
Protein Sequence Analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Human Ku autoantigen binds cisplatin-damaged DNA but fails to stimulate human DNA-activated protein kinase. / Turchi, John; Henkels, Karen.

In: Journal of Biological Chemistry, Vol. 271, No. 23, 1996, p. 13861-13867.

Research output: Contribution to journalArticle

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abstract = "We have identified a series of proteins based on an affinity for cisplatin-damaged DNA. One protein termed DRP-1 has been purified to homogeneity and was isolated as two distinct complexes. The first complex is a heterodimer of 83- and 68-kDa subunits, while the second complex is a heterotrimer of 350-, 83-, and 68-kDa subunits in a 1:1:1 ratio. The 83- and 68-kDa subunits in each complex are identical. The 83-kDa subunit of DRP-1 was identified as the p80 subunit of Ku autoantigen by N-terminal protein sequence analysis and reactivity with a monoclonal antibody directed against human Ku p80 subunit. The 68-kDa subunit of DRP-1 cross-reacted with monoclonal antisera raised against the Ku autoantigen p70 subunit. The 350- kDa subunit was identified as DNA-PK(cs), the catalytic subunit of the human DNA-activated protein kinase, DNA-PK. DRP-1/Ku DNA binding was assessed in mobility shift assays and competition binding assays using cisplatin-damaged DNA. Results indicate that DNA binding was essentially unaffected by cisplatin-DNA adducts in the presence or absence of DNA-PK(cs). DNA-PK activity was only stimulated with undamaged DNA, despite the ability of Ku to bind to cisplatin-damaged DNA. The lack of DNA-PK stimulation by cisplatin- damaged DNA correlated with the extent of cisplatin-DNA adduct formation. These results demonstrate that Ku can bind cisplatin-damaged DNA but fails to activate DNA-PK. These results are discussed with respect to the repair of cisplatin-DNA adducts and the role of DNA-PK in coordinating DNA repair processes.",
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