The potent environmental carcinogen benzo[a]pyrene (B[a]P) is metabolically activated in cells to (+)-anti- B[a]PDE, which forms one primary adduct: [+ta]-B[a]P-N2-dG. [+ta]-B[a]P-N2-dG induces different mutational patterns depending on sequence context (e.g., >95% G->T vs. 95% G->A in 5'-TGC vs. 5'-AGA sequences). Evidence suggests that these different mutations arise from different adduct conformations (as influenced by sequence context), when bypassed by different DNA polymerases. For [+ta]-B[a]P-N2-dG, we showed that E. coli DNA Pol V is involved in dATP bypass (G->T mutations), while dCTP insertion (no mutation) involves Pols IV and V. With the mirror image adduct [-ta]-B[a]P-N2-dG, Pol V does dATP insertion, while Pol IV alone is required for dCTP bypass. Literature findings suggest that in general DNA Pol V has two modes of adduct bypass: (1) correct dNTP insertion, and (2) default dATP insertion. Understanding the mechanism of correct vs. mutagenic insertion is hampered by no X-ray structure for UmuC, which is the the polymerase subunit of DNA Pol V. Using homology modeling, we constructed a UmuC model, which revealed active site amino acids potentially involved in dictating dNTP insertion. Active site amino acids were changed. In cells we showed that mutant-UmuCs could increase (up to ~10-fold), or decrease (~5-fold) dATP insertion compared to wt-UmuC. The goal of this project is to understand what amino acid residues define correct (dCTP) vs. incorrect (dATP) insertion for Pols IV and V and how these pathways are controlled by the cell. Studies in cells and in vitro with mutant and wild type Pols IV and V are proposed. Literature findings show that Pol IV is equivalent to human Pol k, while Pol V is equivalent to human Pol h.
Aim 1. Establish the roles of Pol IV vs. Pol V in cells;i.e., which does insertion vs. extension.
Aim 2 : Determine what amino acids in Pols IV and V control correct (dCTP) vs incorrect (dATP) insertion;e.g., why does Pol IV do correct (dCTP) insertion, while Pol V does incorrect (dATP) insertion, with [-ta]-B[a]P-N2-dG.
Aim 3 : Determine what lesion-bypass Pols are involved in G->A mutagenesis (dTTP insertion).

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES003775-21
Application #
7738480
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Shaughnessy, Daniel
Project Start
1985-06-30
Project End
2011-11-30
Budget Start
2009-12-01
Budget End
2010-11-30
Support Year
21
Fiscal Year
2010
Total Cost
$454,663
Indirect Cost
Name
Boston University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
049435266
City
Boston
State
MA
Country
United States
Zip Code
02215
Sholder, Gabriel; Loechler, Edward L (2015) A method to accurately quantitate intensities of (32)P-DNA bands when multiple bands appear in a single lane of a gel is used to study dNTP insertion opposite a benzo[a]pyrene-dG adduct by Sulfolobus DNA polymerases Dpo4 and Dbh. DNA Repair (Amst) 25:97-103
Sholder, Gabriel; Creech, Amanda; Loechler, Edward L (2015) How Y-Family DNA polymerase IV is more accurate than Dpo4 at dCTP insertion opposite an N2-dG adduct of benzo[a]pyrene. DNA Repair (Amst) 35:144-53
Ikeda, Mio; Furukohri, Asako; Philippin, Gaelle et al. (2014) DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts. Nucleic Acids Res 42:8461-72
Chandani, Sushil; Loechler, Edward L (2013) Structural model of the Y-Family DNA polymerase V/RecA mutasome. J Mol Graph Model 39:133-44
Chandani, Sushil; Loechler, Edward L (2009) Y-Family DNA polymerases may use two different dNTP shapes for insertion: a hypothesis and its implications. J Mol Graph Model 27:759-69
Seo, Kwang Young; Yin, Jun; Donthamsetti, Prashant et al. (2009) Amino acid architecture that influences dNTP insertion efficiency in Y-family DNA polymerase V of E. coli. J Mol Biol 392:270-82
Clapp, Richard W; Jacobs, Molly M; Loechler, Edward L (2008) Environmental and occupational causes of cancer: new evidence 2005-2007. Rev Environ Health 23:1-37
Herscovitch, Melanie; Comb, William; Ennis, Thomas et al. (2008) Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347. Biochem Biophys Res Commun 367:103-8
Chandani, Sushil; Loechler, Edward L (2007) Molecular modeling benzo[a]pyrene N2-dG adducts in the two overlapping active sites of the Y-family DNA polymerase Dpo4. J Mol Graph Model 25:658-70
Kalam, M Abul; Haraguchi, Kazuhiro; Chandani, Sushil et al. (2006) Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells. Nucleic Acids Res 34:2305-15

Showing the most recent 10 out of 41 publications