The broad long-term objective of the proposed research is to further our understanding of the fidelity mechanisms utilized by Pol beta. DNA Polymerase beta (Pol beta) is an enzyme that is known to function in base excision repair (BER) and meiosis. The BER system is responsible for the repair of at least 10,000 lesions per cell per day. Because Pol ? inserts an incorrect nucleotide once for every 10,000 bases it copies, Pol beta has the opportunity to commit 1 error per cell per day during the filling of small gaps resulting from the excision of lesions. These mutations have the potential to result in human disease, including cancer. We will take a combined genetic, biochemical, and structural approach to understand how Pol beta chooses the correct nucleotide substrate for incorporation into DNA.
The Specific Aims are: (1) To test the hypothesis that distinct structural sub-domains of Pol beta that we identified during the last funding period are critical for fidelity; (2) To determine if structural sub-domains or amino acid residues of Pol beta other than the ones we have identified previously are critical for fidelity and to understand how they contribute Ito this process; (3) To test the hypothesis that alteration of distinct structural subdomains within Pol beta results in a mutator phenotype in mouse cells. The results of the proposed mechanistic studies of Pol beta will further our understanding of the molecular basis of mutation and have the potential to contribute directly to our understanding of the etiology of significant human diseases, including cancer. Our approach includes identifying and characterizing DNA Polymerase beta mutants using a genetic screen we developed, and analyzing these mutants biochemically and structurally. We also will characterize the phenotypes of Polymerase beta mutants in vivo.
|Oertell, Keriann; Kashemirov, Boris A; Negahbani, Amirsoheil et al. (2018) Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State. Biochemistry 57:3925-3933|
|Liptak, Cary; Mahmoud, Mariam M; Eckenroth, Brian E et al. (2018) I260Q DNA polymerase ? highlights precatalytic conformational rearrangements critical for fidelity. Nucleic Acids Res 46:10740-10756|
|Huang, Ji; Alnajjar, Khadijeh S; Mahmoud, Mariam M et al. (2018) The nature of the DNA substrate influences pre-catalytic conformational changes of DNA polymerase ?. J Biol Chem 293:15084-15094|
|Mahmoud, Mariam M; Schechter, Allison; Alnajjar, Khadijeh S et al. (2017) Defective Nucleotide Release by DNA Polymerase ? Mutator Variant E288K Is the Basis of Its Low Fidelity. Biochemistry 56:5550-5559|
|Eckenroth, Brian E; Towle-Weicksel, Jamie B; Nemec, Antonia A et al. (2017) Remote Mutations Induce Functional Changes in Active Site Residues of Human DNA Polymerase ?. Biochemistry 56:2363-2371|
|Alnajjar, Khadijeh S; Garcia-Barboza, Beatriz; Negahbani, Amirsoheil et al. (2017) A Change in the Rate-Determining Step of Polymerization by the K289M DNA Polymerase ? Cancer-Associated Variant. Biochemistry 56:2096-2105|
|Alnajjar, Khadijeh S; Negahbani, Amirsoheil; Nakhjiri, Maryam et al. (2017) DNA Polymerase ? Cancer-Associated Variant I260M Exhibits Nonspecific Selectivity toward the ?-? Bridging Group of the Incoming dNTP. Biochemistry 56:5449-5456|
|Sohl, Christal D; Ray, Sreerupa; Sweasy, Joann B (2015) Pools and Pols: Mechanism of a mutator phenotype. Proc Natl Acad Sci U S A 112:5864-5|
|Towle-Weicksel, Jamie B; Dalal, Shibani; Sohl, Christal D et al. (2014) Fluorescence resonance energy transfer studies of DNA polymerase ?: the critical role of fingers domain movements and a novel non-covalent step during nucleotide selection. J Biol Chem 289:16541-50|
|Eckenroth, Brian E; Fleming, Aaron M; Sweasy, Joann B et al. (2014) Crystal structure of DNA polymerase ? with DNA containing the base lesion spiroiminodihydantoin in a templating position. Biochemistry 53:2075-7|
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