title: Increased flexibility enhances misincorporation: temperature effects on nucleotide incorporation opposite a bulky carcinogen-DNA adduct by a Y-family DNA polymerase.
abstract: The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55 degrees C and results compared with an earlier study at 37 degrees C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55 degrees C compared with 37 degrees C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55 degrees C compared with 37 degrees C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the kcat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.
authors.affiliation: Department of Biology, New York University, New York, New York 10003, USA.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Perlow-Poehnelt
authors.forename: Ilya
authors.initials: I
authors.lastname: Likhterov
authors.forename: Lihua
authors.initials: L
authors.lastname: Wang
authors.forename: David A
authors.initials: DA
authors.lastname: Scicchitano
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Geacintov
authors.forename: Suse
authors.initials: S
authors.lastname: Broyde
mesh_terms: D020137:Base Pair Mismatch; D001564:Benzo(a)pyrene / Q000737:chemistry*; D002273:Carcinogens / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry*; D019951:DNA Polymerase beta / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides / Q000235:genetics; D011487:Protein Conformation; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics*
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D009711:Nucleotides; D001564:Benzo(a)pyrene; D006147:Guanine; D019951:DNA Polymerase beta