Cancer-causing substances or \u201ccarcinogens\u201d 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 \u201cpolycyclic aromatic hydrocarbon\u201d 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 \u201cBP-dG\u201d ([+ta]-B[a]P-N2-dG), which causes many kinds of mutations, notably G->T and G->C.<\/p>\n
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.<\/p>\n
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.<\/p>\n
Thus, our work is revealing how Y-Family DNAPs are accurate in some cases, but cause mutations relevant to cancer causation in other cases.<\/p>\n