Double strand breaks, which can be introduced in DNA by a variety of environmental insults including ionizing radiation and exposure to chemical damage, can cause mutations and major genetic rearrangements. This project will use a model DNA replication system based upon bacteriophage T7 infected Escherichia coli to study the mechanisms by which double strand breaks are repaired. Double strand breaks will be placed at well defined sites on T7 genomes and their repair monitored by restoration of intact T7 genomes as determined either by packaging the DNA or by physical assay. One series of experiments will seek to determine why repair of a double strand break is often accompanied by acquisition of genetic material from an intact genome present in the same reaction mixtures. DNA replication accompanying repair of double strand breaks will be measured. The role of the intact DNA molecules in the repair process will be determined by looking for physical exchange between DNA molecules and by determining whether the intact genomes serve primarily as a template for extension of the broken partial genomes. A second objective is to determine why when double strand form between directly repeated sequences repair of the breaks are often accompanied by deletion of the DNA between the repeats. Experiments will establish if this repair mechanism involves annealing between exposed single stand copies of the repeats or whether direct ligation of the broken ends contributes to the repair. The roles of specific T7 and E. coli proteins in repair will be investigated. The intent of these experiments is to derive a more complete understanding of the process by which double strand breaks are repaired by using a relatively simple DNA replication system that is highly amenable to experimentation. Extrapolation from these studies will provide valuable guidance for studies with more complex systems.
|Yu, M; Masker, W (2001) T7 single strand DNA binding protein but not T7 helicase is required for DNA double strand break repair. J Bacteriol 183:1862-9|
|Lai, Y T; Masker, W (2000) Repair of double-strand breaks by incorporation of a molecule of homologous DNA. Mol Microbiol 36:437-46|
|Lai, Y T; Masker, W (2000) Visualization of repair of double-strand breaks in the bacteriophage T7 genome without normal DNA replication. J Bacteriol 182:327-36|
|Lai, Y T; Masker, W (1998) In vitro repair of gaps in bacteriophage T7 DNA. J Bacteriol 180:6193-202|