The long-term goal of this research program is to develop an integrated mechanistic view of how organisms coordinate the actions of their replication machinery with those of other cellular factors involved in DNA repair and damage tolerance. Failure to do so leads to a loss of genetic fidelity and contributes to human disease. Work from our laboratory and others have demonstrated unambiguously that DNA polymerase (Pol) processivity clamps (? or DnaN sliding clamps) play multiple essential roles in this highly complex process. The proposed research program utilizes an integrated genetic-biochemical-physical biochemical approach, placing particular emphasis on determining how the ? clamp coordinates the actions of the E. coli replicase, DNA polymerase III holoenzyme (Pol III HE), with the polB-encoded Pol II and the dinB-encoded Pol IV, which act in replication and translesion DNA synthesis (TLS), as well as with the Hda protein, which regulates initiation of DNA replication by inactivating the DnaA initiator protein. Over the next progress period, we will utilize in vitro assays to characterize interactions of Pol III HE, Pol II, and Pol IV with various mutant ? clamp proteins. As part of this work, we will purify heterodimeric clamp proteins bearing either a single mutation in one subunit, or different mutations in each subunit. Using these mutant clamps, we will dissect the mechanism(s) by which the ? clamp mediates Pol switching to coordinate high fidelity replication with TLS. We will also utilize genetic approaches to define the mechanism(s) of Pol switching in vivo, and to determine whether additional cellular factors contribute to this critically important process. We anticipate that model(s) for Pol switching supported by our results will serve as a valuable paradigm for similar switch mechanisms in other organisms, including humans. Moreover, since TLS Pols are well conserved throughout all three branches of life, results from our studies will also contribute to our understanding of the mechanisms underlying mutagenesis under times of stress, thereby impacting on pathogenesis and antibiotic resistance, as well as the mechanism(s) by which TLS Pols contribute to immunoglobulin diversity during somatic hypermutation. We will also apply the approaches that we are developing to characterize Pol switching to the Hda protein in order to define the mechanism by which E. coli coordinates replication with Hda-dependent regulation of initiation of replication. Failure to properly regulate initiation can be lethal. We will distinguish between different models for Hda function, and will determine whether Hda and Pol III HE simultaneously bind to the same ? clamp. We will also utilize genetic and biochemical approaches to determine whether Hda acts to regulate access of TLS Pols to the replication fork until such time as they are required. Since replication errors contribute significantly to mutagenesis, and since the coordinate regulation of initiation and elongation of DNA replication is critically important for genome stability, our findings in these areas may also identify new classes of targets for the development of novel antibiotics.

Public Health Relevance

Failure to coordinate the actions of the different replication and repair factors leads to a loss of genetic fidelity and contributes to human disease. Since mechanisms of replication and repair are remarkably well conserved from bacteria to humans, we will utilize Escherichia coli as a model system to understand how the actions of different replication and repair factors are coordinately regulated with each other. We anticipate that our results will serve as a framework for understanding similar control networks in humans, were the complexity of the events is far greater, and as such, will contribute to our understanding of mechanisms contributing to cancer and other human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066094-09
Application #
8269036
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Janes, Daniel E
Project Start
2003-05-01
Project End
2013-09-26
Budget Start
2012-06-01
Budget End
2013-09-26
Support Year
9
Fiscal Year
2012
Total Cost
$297,976
Indirect Cost
$89,705
Name
State University of New York at Buffalo
Department
Biochemistry
Type
Schools of Medicine
DUNS #
038633251
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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