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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066094-11
Application #
8744275
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Willis, Kristine Amalee
Project Start
2003-05-01
Project End
2017-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
11
Fiscal Year
2014
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Nanfara, Michael T; Babu, Vignesh M P; Ghazy, Mohamed A et al. (2016) Identification of β Clamp-DNA Interaction Regions That Impair the Ability of E. coli to Tolerate Specific Classes of DNA Damage. PLoS One 11:e0163643
Yuan, Quan; Dohrmann, Paul R; Sutton, Mark D et al. (2016) DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a Ï„-Containing DnaX Complex to Enable Exchange into Replication Forks. J Biol Chem 291:11727-35
Ly, Neang S; Bulman, Zackery P; Bulitta, Jürgen B et al. (2016) Optimization of Polymyxin B in Combination with Doripenem To Combat Mutator Pseudomonas aeruginosa. Antimicrob Agents Chemother 60:2870-80
Kath, James E; Chang, Seungwoo; Scotland, Michelle K et al. (2016) Exchange between Escherichia coli polymerases II and III on a processivity clamp. Nucleic Acids Res 44:1681-90
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
Sutton, Mark D (2015) How MutS finds a needle in a haystack. Proc Natl Acad Sci U S A 112:15265-6
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
Scotland, Michelle K; Heltzel, Justin M H; Kath, James E et al. (2015) A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis. PLoS Genet 11:e1005507
Kath, James E; Jergic, Slobodan; Heltzel, Justin M H et al. (2014) Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis. Proc Natl Acad Sci U S A 111:7647-52
Babu, Vignesh M P; Sutton, Mark D (2014) A dnaN plasmid shuffle strain for rapid in vivo analysis of mutant Escherichia coli β clamps provides insight into the role of clamp in umuDC-mediated cold sensitivity. PLoS One 9:e98791

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