Differential activation of DNA-PK based on DNA strand orientation and sequence bias

Katherine S. Pawelczak, Brooke J. Andrews, John J. Turchi

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

DNA-PKcs and Ku are essential components of the complex that catalyzes non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Ku, a heterodimeric protein, binds to DNA ends and facilitates recruitment of the catalytic subunit, DNA-PKcs. We have investigated the effect of DNA strand orientation and sequence bias on the activation of DNA-PK. In addition, we assessed the effect of the position and strand orientation of cisplatin adducts on kinase activation. A series of duplex DNA substrates with site-specific cisplatin-DNA adducts placed in three different orientations on the duplex DNA were prepared. Terminal biotin modification and streptavidin (SA) blocking was employed to direct DNA-PK binding to the unblocked termini with a specific DNA strand orientation and cisplatin-DNA adduct position. DNA-PK kinase activity was measured and the results reveal that DNA strand orientation and sequence bias dramatically influence kinase activation, only a portion of which could be attributed to Ku-DNA binding activity. In addition, cisplatin-DNA adduct position resulted in differing degrees of inhibition depending on distance from the terminus as well as strand orientation. These results highlight the importance of how local variations in DNA structure, chemistry and sequence influence DNA-PK activation and potentially NHEJ.

Original languageEnglish (US)
Pages (from-to)152-161
Number of pages10
JournalNucleic acids research
Volume33
Issue number1
DOIs
StatePublished - 2005

ASJC Scopus subject areas

  • Genetics

Fingerprint Dive into the research topics of 'Differential activation of DNA-PK based on DNA strand orientation and sequence bias'. Together they form a unique fingerprint.

Cite this