Failure to efficiently coordinate DNA replication with other cellular processes results in mutations and genome instability, contributing to numerous human disease states, including cancers. Mutations in human pathogens, particularly those caused by reactive oxygen species (ROS) generated by the host immune response, or exposure to antibiotics, promote their adaptation to the host (i.e., pathoadaptation), exacerbating treatment. The long-term goal of our research is to develop an integrated mechanistic understanding of how organisms coordinate the actions of their DNA replication machinery with those of other cellular factors that act in DNA repair and damage tolerance. Work in our lab over the last 10 years supported by this grant has had a major impact on our understanding of mechanisms coordinating the actions of the E. coli replicase with those of translesion DNA synthesis DNA polymerases (TLS Pols). Our findings successfully challenged the well- established tool belt model. We have also shown that errors catalyzed by Pseudomonas aeruginosa DNA polymerase IV (Pol IV) contribute to mutations that likely promote persistence of this pathogen in cystic fibrosis airways. A molecular understanding of the mechanisms that contribute to mutations is crucial to our understanding of the basis for genome instability, human disease, and pathoadaptation, as well as efforts to develop novel therapies. The proposed research addresses unanswered questions regarding mechanisms that organisms use to manage the actions of their diverse Pols. We will focus our efforts in two critical yet understudied areas. During the prior period of support, we discovered that specific E. coli beta-clamp-DNA interactions are required for DNA damage-induced mutagenesis, suggesting they impart a hierarchical order to Pol switches that may be exploited to control mutation rate.
In Aim 1, we will determine the contributions of the different beta-clamp-DNA interactions to replication fidelity and TLS using a combination of genetic, biochemical, biophysical, and single molecule approaches.
In Aim 2, we will use small angle X-ray scattering (SAXS), size exclusion chromatography-multi angle light scattering (SEC-MALS), molecular modeling, and biochemical approaches to structurally define complexes consisting of the 5 different E. coli Pols, clamp, and DNA. Using insights gained from these efforts, together with genetic, biochemical, biophysical, and single molecule approaches, we will define the mechanisms by which E. coli Pols switch. We will also determine whether an ability to impede Pol III processivity is shared by other proteins that switch with Pol III. Results from these experiments will provide unprecedented insight into the molecular mechanisms underlying coordinate regulation of DNA replication, DNA repair, and TLS. Furthermore, we anticipate that our results will identify critical steps in these evolutionarily conserved processes that can be targeted to control proficiency and fidelity of replication for therapeutic gain.

Public Health Relevance

Mutations contribute directly to human diseases, including cancers;they also complicate treatment of individuals infected with pathogenic bacteria by conferring antibiotic resistance and adaptation of these pathogens to their human host. We will define mechanisms by which low fidelity bacterial DNA polymerases gain access to the replication fork to catalyze mutations. We anticipate that our results will identify critical stepsin these control networks that may be targeted to control replication and mutagenesis for therapeutic gain, and will also serve as a framework for understanding similar control networks in humans, contributing to our understanding of mechanisms underlying human diseases such as cancer, as well as aging.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Willis, Kristine Amalee
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State University of New York at Buffalo
Schools of Medicine
United States
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Kim, Jin S; Nanfara, Michael T; Chodavarapu, Sundari et al. (2017) Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing. Nucleic Acids Res 45:3888-3905
Babu, Vignesh M P; Itsko, Mark; Baxter, Jamie C et al. (2017) Insufficient levels of the nrdAB-encoded ribonucleotide reductase underlie the severe growth defect of the ?hda E. coli strain. Mol Microbiol 104:377-399
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Pillon, Monica C; Babu, Vignesh M P; Randall, Justin R et al. (2015) The sliding clamp tethers the endonuclease domain of MutL to DNA. Nucleic Acids Res 43:10746-59
Bulman, Zackery P; Sutton, Mark D; Ly, Neang S et al. (2015) Emergence of polymyxin B resistance influences pathogenicity in Pseudomonas aeruginosa mutators. Antimicrob Agents Chemother 59:4343-6
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