Stable down-regulation of human polynucleotide kinase enhances spontaneous mutation frequency and sensitizes cells to genotoxic agents
Abstract
Human polynucleotide kinase (hPNK) is a 57.1-kDa monomeric protein with conserved motifs associated with phosphatase and kinase activities. hPNK catalyzes phosphorylation of 5'-DNA termini and dephosphorylation of 3'-DNA termini. Previous studies, employing cell-free systems, have suggested that hPNK participates in the repair of DNA strand breaks. To better define the cellular function of hPNK, a double-stranded small-interfering RNA molecule designed to stably target hPNK transcription was introduced into A549 human lung adenocarcinoma cells. The small-interfering RNA suppressed hPNK gene expression by at least 80-90%. These cells exhibited a 7-fold higher spontaneous mutation frequency based on the development of resistance to ouabain; elevated sensitivity to a broad range of genotoxic agents including γ-radiation, UVC radiation, methyl methanesulfonate, hydrogen peroxide, and camptothecin; and slower repair of radiation-induced DNA strand breaks. These findings underscore the importance of hPNK in the maintenance of DNA integrity after damage induced by endogenous and exogenous agents.
- Publication:
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Proceedings of the National Academy of Science
- Pub Date:
- May 2004
- DOI:
- 10.1073/pnas.0400099101
- Bibcode:
- 2004PNAS..101.6905R
- Keywords:
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- Biochemistry