Genomic Maintenance & Repair
Monday, October 17, 2005 3:00 PM-5:00 PM Exhibit Hall(PP363) Cellular evidence supporting human polynucleotide kinase involvement in the non-homologous end joining DNA repair pathway.
Karimi-Busheri, Feridoun1, Rasouli-Nia, Aghdass1, Allalunis-Turner, Joan1, Weinfeld, Michael*,1, 1 Experimental Oncology, Edmonton, Alberta, Canada
ABSTRACT- DNA double-strand breaks (DSBs) contribute significantly towards genomic instability and cell lethality. As a consequence, eukaryotic cells have developed several pathways to rejoin DNA at such breaks, the major ones being homologous recombination and non-homologous end joining (NHEJ). Many DSBs require end-processing of their termini before strand rejoining can be completed. DNA ligases, in particular, require 5′-phosphate and 3′-hydroxyl DNA termini for strand rejoining. One enzyme that generates such termini is human polynucleotide kinase (hPNK), which catalyzes phosphorylation of 5′-DNA termini and dephosphorylation of 3′-DNA termini. Although there is evidence indicating a role for PNK in the NHEJ pathway, all the data to date have been obtained through in vitro studies using purified proteins, including XRCC4, or cell extracts. We sought further evidence for direct involvement of hPNK in the NHEJ pathway using a cell-based system to compare the influence of hPNK down-regulation on the response of NHEJ-proficient and deficient cells (MO59K and MO59J glioblastoma cells, respectively) to ionizing radiation. Expression of hPNK was stably down-regulated in these cells by expressing an anti-hPNK RNAi using the pSuper vector. We observed that while reduced expression of hPNK in the NHEJ-proficient cell lines increased cellular sensitivity to ionizing radiation, it had no effect on the response of the NHEJ-deficient cell line. This result was also reflected in the repair of DSB as judged by histone H2AX phosphorylation. To judge potential involvement of hPNK in homologous recombination, we examined the induction of sister chromatid exchanges (SCE) following irradiation of repair proficient cells and cells with reduced hPNK expression. No difference in SCE induction was observed. These data suggest that hPNK participates in the NHEJ pathway but not in homologous recombination.
Key words: DNA repair, double strand breaks, DNA termini