Genetic analysis of homologous recombination repair of DNA double-strand breaks in Drosophila melanogaster.
Abstract: Eukaryotic cells employ a number of pathways to ensure that their
genomes are faithfully duplicated and accurately repaired when damaged. One critical
pathway for the repair of DNA double strand breaks (DSBs) is homologous recombination (HR).
HR involves copying DNA sequence from an intact template, often a sister chromatid, to
restore genetic information lost at a DSB site. The HR ... read morepathway is dependent on many
proteins, including BRCA2, RAD51, HELQ and BLM. Mutations in these and other repair
proteins result in an increased risk for various cancers, but their molecular mechanisms
are not completely understood. We have used Drosophila melanogaster to investigate various
aspects of HR using a genetic approach. First, we confirmed that Drosophila Brca2 and Rad51
are equally important in the repair of DSBs induced by transposon excision, mutagenic
chemicals or radiation. This is consistent with the established role for Brca2 in
recruiting Rad51 to the sites of DSBs. Unlike its mammalian homologs, DmBrca2 does not have
a Rad51-independent role in mitotic DSB repair. We went on to show that brca2 mutants in
flies are sensitive to the topoisomerase inhibitor, camptothecin. This sensitivity is
exacerbated by mutations in the cytochrome P450 gene, Cyp6d2. We showed that cyp6d2
mutations are widespread in Drosophila stocks, which can skew results from experiments
using this drug to investigate DNA repair mechanisms. We rescued the sensitivity phenotype
using a wild type copy of Cyp6d2. We also looked at the steps of HR downstream of Rad51
recruitment. We showed that DmHelQ and DmBlm are both involved in promoting accurate HR
repair of a DSB. In the absence of either or both of these enzymes, we see a decrease in
the frequency and efficiency of accurate repair along with a compensatory increase in
inaccurate repair processes. We suggest a model to explain these observations. Together,
our data have clarified several intermediate steps of homologous recombination repair of a
DSB. This dissertation illustrates the effectiveness of using Drosophila melanogaster to
study DNA repair mechanisms.
Thesis (Ph.D.)--Tufts University, 2013.
Submitted to the Dept. of Biology.
Advisor: Mitch McVey.
Committee: Catherine Freudenreich, Sergei Mirkin, Susan Ernst, and Michael Brodsky.
Keyword: Biology.read less