Replication fork repair is essential for cell survival and stability of genetic material. In humans, inefficiency of repair is associated with cancer proneness, neurological, immunological, developmental defects and premature aging. The long-term goal of this study is a more complete mechanistic understanding of the repair of replication gaps. The objective of this project will be to elucidate elements of replication gap repair pathways, including specific replisome components, using genetic and biochemical experiments. Because all cells repair DNA in fundamentally similar ways by evolutionarily related pathways, these studies using the model organism, Escherichia coli, should reveal mechanisms applicable to repair of DNA in human cells. The use of azidothymidine (AZT) in this study as a compound to induce replication gaps has the potential to reveal toxicity and tolerance mechanisms relevant to its therapeutical use as an anti-HIV agent. In addition, because the DNA damage response in bacterial pathogens plays a role in toxin production, antibiotic resistance and persistence of infection, this work could provide new targets for antibiotic therapy.
The first aim of this proposal is to identify the mechanism by which RadA, a universal bacterial protein related to RecA, assists homologous recombination. In vitro, RadA stimulates RecA-mediated recombination reactions by accelerating branch migration of strand exchange intermediates. The mechanism of this stimulation will be deduced by biochemical and genetic experiments, to determine how RadA and RecA interact to promote recombination.
The second aim of this study seeks to clarify how replisome clamp-loader complexes and repair proteins interact to facilitate repair. The YoaA protein, a member of the XPD/FANCJ family of helicases mutated in human disease syndromes, will be studied by genetic and biochemical methods to ascertain whether it is a DNA helicase, how this activity is specialized and how it promotes replication gap repair. Interactions with the replisome will be examined genetically and biochemically. The abundance, in vivo, of DnaX-variant clamp- loader complexes and their regulation will be determined, as well as their role in promoting genetic stability. A new method for isolation of proteins associated with AZT-terminated gaps will be developed to identify and assay recruitment of replication and repair proteins to sites of replication blockage. This study will significantly advance the field of DNA repair by providing new information regarding how replication gaps are sensed and repaired.
|Lovett, Susan T (2017) Template-switching during replication fork repair in bacteria. DNA Repair (Amst) 56:118-128|
|Cooper, Deani L; Lovett, Susan T (2016) Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli. Elife 5:|
|Cooper, Deani L; Boyle, Daniel C; Lovett, Susan T (2015) Genetic analysis of Escherichia coli?RadA: functional motifs and genetic interactions. Mol Microbiol 95:769-79|
|Brown, Laura T; Sutera Jr, Vincent A; Zhou, Shen et al. (2015) Connecting Replication and Repair: YoaA, a Helicase-Related Protein, Promotes Azidothymidine Tolerance through Association with Chi, an Accessory Clamp Loader Protein. PLoS Genet 11:e1005651|
|Anand, Ranjith P; Lovett, Susan T; Haber, James E (2013) Break-induced DNA replication. Cold Spring Harb Perspect Biol 5:a010397|
|Seier, Tracey; Zilberberg, Gal; Zeiger, Danna M et al. (2012) Azidothymidine and other chain terminators are mutagenic for template-switch-generated genetic mutations. Proc Natl Acad Sci U S A 109:6171-4|
|Seier, Tracey; Padgett, Dana R; Zilberberg, Gal et al. (2011) Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events. Genetics 188:247-62|
|Lovett, Susan T (2011) The DNA Exonucleases of Escherichia coli. EcoSal Plus 4:|
|Cooper, Deani L; Lovett, Susan T (2011) Toxicity and tolerance mechanisms for azidothymidine, a replication gap-promoting agent, in Escherichia coli. DNA Repair (Amst) 10:260-70|
|Merrikh, Houra; Ferrazzoli, Alexander E; Lovett, Susan T (2009) Growth phase and (p)ppGpp control of IraD, a regulator of RpoS stability, in Escherichia coli. J Bacteriol 191:7436-46|
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