Using a modified mobility shift assay, we have found find that DNA-ends will bind preferentially to circular plasmid DNA containing matrix attachment region (MAR) DNA sequences and that this interaction requires the DNA-end-binding protein Ku, DNA dependent protein kinase catalytic subunit (DNA-PKcs), and other unknown factors in nuclear extract. A biochemical screen identified a protein fraction containing a major band of 55 kD and one other band of at least 10 fold intensity which stimulated plasmid DNA-end binding when added with purified Ku/DNA-PKcs. Amino acid sequencing of this protein identified a human cDNA sequence which codes for a 55 kD protein that similar to the yeast Saccharomyces cerevisiae DNA repair gene PSO4. We hypothesize that the human homolog of the yeast PSO4 protein, bPSO4, functions in the repair of double strand DNA breaks and interacts with DNA-PKcs/Ku to direct the association of DNA ends with MAR DNA sequences on the nuclear matrix.
Aim 1 is to Investigate the involvement of the human PSO4 protein in DNA-PKcS/Ku mediated association of DNA ends with MAR DNA sequences and identify other proteins that may be involved with PSO4 protein in this association. The ability of purified recombinant hPSO4 protein to stimulate binding of DNA-ends to MAR plasmid DNA by purified DNA-PKcs/Ku will be assayed. Poly- and monoclonal antibodies to the hPSO4 protein will be prepared and used to co-immunoprecipitate and identify hPSO4-associated proteins from nuclear extracts prepared from DNA-damaged and non-damaged cells. Monoclonal HPSO4 antibody will be used to determine whether the distribution of the protein changes in response to DNA damage and whether the hPSO4 protein co-localizes with other DSB repair proteins (DNA-PK/Ku, XRCC4, ligase IV).
In Aim 2 we will explore the possibility of obtaining cell lines that are deficient in the expression of PSO4 protein. Targeted gene disruption and/or expression of antisense hPSO4 mRNA will be used generate cell lines defective in hPSO4 protein expression. These lines will be assayed for increased sensitivity to DNA damaging agents and their phenotype compared to the yeast pso4-1 mutant. We believe that understanding the repair function of the human P04 homolog will shed new light on the mechanism of double strand DNA break repair in human cells and help us understand the role of this repair pathway in genetic diseases like cancer.
| Lorenzini, Antonello; Johnson, F Brad; Oliver, Anthony et al. (2009) Significant correlation of species longevity with DNA double strand break recognition but not with telomere length. Mech Ageing Dev 130:784-92 |
