Cancer-causing substances or “carcinogens” cause mutations, which makes sense, since tumor cells have mutations in key genes involved in growth control. Benzo[a]pyrene (B[a]P) is a potent mutagen-carcinogen in the “polycyclic aromatic hydrocarbon” class, which are ubiquitous environmental contaminants produced by incomplete combustion (e.g., in car exhaust, power plant emissions, cigarette smoke, and charred foods). B[a]P is often the most important component of soot based on its prevalence and its potency. B[a]P reacts at N2-guanine to give “BP-dG” ([+ta]-B[a]P-N2-dG), which causes many kinds of mutations, notably G->T and G->C.
We investigate bypass of BP-dG by DNA polymerases (DNAPs), which principally involves Y-Family DNAPs. In human cells DNAP kappa correctly inserts dCTP, while DNAP eta incorrectly misinserts dATP and dGTP. To more readily investigate how related Y-Family DNAPs bypass BP-dG so differently, we use purified E. coli DNAP IV, which also correctly inserts dCTP, and Sulfolobus solfataricus Dpo4, which also misinserts dATP and dGTP.
By making mutations in DNAP IV and Dpo4 that affect dCTP insertion rate or dATP/dGTP misinsertion rate, we have identified two protein structural elements in Y-Family DNAPs that contribute to the fidelity of insertion opposite BP-dG, (1) To form BP-dG:dCTP Watson-Crick pairing, the BP-moiety must be on the minor groove of the active site, where a large opening in the protein surface enhances the rate of correct dCTP insertion. DNAP IV has a large minor groove opening, while Dpo4 does not. (2) Y-Family DNAPs form non-covalent bridges (NCB) between their thumb/palm/fingers-domains (TPF-domains), which operate as a unit, and their little finger domain (LF-domain). The quantity and quality of these NCBs suppress incorrect dGTP/dATP misinsertion. We have also shown that the BP-moiety is on the major groove side of the active site during dGTP misinsertion, but on the minor grove side during dATP misinsertion.
Thus, our work is revealing how Y-Family DNAPs are accurate in some cases, but cause mutations relevant to cancer causation in other cases.
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- Chandani S, Jacobs C, Loechler EL (2010) Architecture of y-family DNA polymerases relevant to translesion DNA synthesis as revealed in structural and molecular modeling studies. Journal of Nucleic Acids. pii: 784081.
- Chandani S, Loechler EL (2010) Translesion synthesis and mutagenic pathways in E. coli cells. The Chemical Biology of DNA Damage, Wiley-VCH, Weinheim, Germany, pp. 353-380.
- Chandani S, Loechler EL (2009) Y-family DNA polymerases may use two different dNTP shapes for insertion: A hypothesis and its implications Journal of Molecular Graphics and Modelling. 27, 759 – 769.
- Seo K-Y, Yin J, Donthamsetti P, Chandani S, Lee CH, Loechler EL (2009) Amino acid architecture that influences dNTP insertion efficiency in Y-family DNA polymerase V of E. coli. Journal of Molecular Biology. 392, 270 – 282.
- Clapp RW, Jacobs MW, Loechler EL (2008) Environmental and occupational causes of cancer: new evidence 2005 – 2007. Rev. Env. Health 23, 1-37.
- Chandani S, Lee CH, Loechler EL (2007) Molecular modeling benzo[a]pyrene N2-dG adducts in two partially overlapping active sites of the y-family DNA polymerase Dpo4. Journal of Molecular Graphics and Modelling. 25, 658-670.
- Seo K -Y, Nagalingam A, Miri S, Yin J, Kolbanovskiy A, Shastry A, Loechler EL (2006) Mirror image stereoisomers of the major benzo[a]pyrene N2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli. DNA Repair 5, 515-527.
- BI216 Intensive Genetics
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