title: Mechanistic insights into replication across from bulky DNA adducts: a mutant polymerase I allows an N-acetyl-2-aminofluorene adduct to be accommodated during DNA synthesis.
abstract: The molecular mechanism that allows a polymerase to incorporate a nucleotide opposite a DNA lesion is not well-understood. One way to study this process is to characterize the altered molecular interactions that occur between the polymerase and a damaged template. Prior studies have determined the polymerase-template dissociation constants and used kinetic analyses and a protease digestion assay to measure the effect of various DNA adducts positioned in the active site of Klenow fragment (KF). Here, a mutator polymerase was used in which the tyrosine at position 766 of the KF has been replaced with a serine. This position is located at the junction of the fingers and palm domain and is thought to be involved in maintaining the active site geometry. The primer-template was modified with N-acetyl-2-aminofluorene (AAF), a well-studied carcinogenic adduct. The mutant polymerase displayed a significant increase in the rate of incorporation of the correct nucleotide opposite the adduct but was much less prone to incorporate an incorrect nucleotide relative to the wild-type polymerase. Both the wild-type and the mutant polymerase bound much more tightly to the AAF-modified primer-template; however, unlike the wild-type polymerase, the binding strength of the mutant was influenced by the presence of a dNTP. Moreover, the mutant polymerase was able to undergo a dNTP-induced conformational change when the AAF adduct was positioned in the active site, while the wild-type enzyme could not. A model is proposed in which the looser active site of the mutant is able to better accommodate the AAF adduct.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
authors.forename: Samer
authors.initials: S
authors.lastname: Lone
authors.forename: Louis J
authors.initials: LJ
authors.lastname: Romano
mesh_terms: D015073:2-Acetylaminofluorene / Q000633:toxicity; D001665:Binding Sites; D002273:Carcinogens / Q000633:toxicity; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage; D004256:DNA Polymerase I / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology*; D005449:Fluorenes / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D013698:Templates, Genetic; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: C052668:2-aminofluorene-DNA complex; D002273:Carcinogens; D018736:DNA Adducts; D017931:DNA Primers; D005449:Fluorenes; D014443:Tyrosine; D015073:2-Acetylaminofluorene; D004256:DNA Polymerase I; D014357:Trypsin