Ionizing Radiation and a number of radiomimetic agents induce DNA double strand breaks (DSB). Cellular mechanisms rejoin these broken ends occasionally resulting in chromosomal aberrations such as translocations. Chromosomal translocations can lead to altered gene expression, transformed cellular phenotype and oncogenesis. The proteins involved in DSB rejoining have not been fully identified; however, a number of proteins such as DNA polymerase, Ku and p350(DNA -pk) have been implicated in the reaction. DNA polymerase has been shown to be involved, late in the end-joining reaction, in filling in single strand gaps. The human Ku heterodimer and p350, components of a DNA dependent protein kinase (DNA-pk) are thought to be involved in DSB rejoining reaction because mutant cells deficient in these factors are also sensitive to ionizing radiation and show decreased levels of DNA end joining. Although these proteins have been implicated in DSB rejoining, their individual roles in DSB rejoining have not been clearly established. The overall goal of this proposal is to identify the factors in mammalian cells involved in rejoining two ends of a DNA double strand break (DSB). Studies using purified exonuclease-free Klenow fragment (KF) demonstrated its capacity to align two ends of a DSB and synthesize DNA across the gap in vitro (King et al. 1994). Furthermore, sequencing of the resultant junctions left by this polymerase showed striking similarities to the mutational spectra seen for restriction enzyme-induced nonhomologous recombination in the APRT locus in Chinese Hamster Ovary (CHO) cells (Phillips and Morgan 1994). 1. Based on this and other evidence (see Preliminary Results), we hypothesize that mammalian DNA polymerase acts to bring two ends of a DSB together. 2. We also hypothesize that a number of other known or unknown factors may interact with DNA polymerase to facilitate DNA end-joining in mammalian cells. 3. Furthermore, we hypothesize that certain features of the DNA ends (e.g. small stretches of sequence homology or mismatches)will affect the efficiency of trans-acting factors to join DNA ends.
| Hopfner, K P; Karcher, A; Shin, D et al. (2000) Mre11 and Rad50 from Pyrococcus furiosus: cloning and biochemical characterization reveal an evolutionarily conserved multiprotein machine. J Bacteriol 182:6036-41 |
| King, J; Fairley, C; Morgan, W (1998) The joining of blunt DNA ends to 3'-protruding single strands in Escherichia coli. Nucleic Acids Res 26:1749-54 |
| Islas, L; Fairley, C F; Morgan, W F (1998) DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination. Nucleic Acids Res 26:3729-38 |
