Effects of base sequence context on translesion synthesis past a bulky (+)-trans-anti-B[a]P-N2-dG lesion catalyzed by the Y-family polymerase pol kappa.
The effects of bases flanking single bulky lesions derived from the binding of a benzo[a]pyrene 7,8-diol 9,10-epoxide derivative ((+)-7R,8S,9S,10R stereoisomer) to N(2)-guanine (G*) on translesion bypass catalyzed by the Y-family polymerase pol kappa (hDinB1) were examined in vitro. The lesions were positioned near the middle of six different 43-mer 5'-...XG*Y... sequences (X, Y = C, T, or G, with all other bases remaining fixed). The complementary dCTP is preferentially inserted opposite G* in all of the sequences; however, the proportions of other dNTPs inserted varies as a function of X and Y. The dCTP insertion efficiencies, f(ins) = (V(max)/K(m))(ins), are smaller in the XG*Y than in XGY sequences by factors of approximately 50-90 (GG*T and GG*C) or 5000-25000 (TG*G and CG*G). Remarkably, in XG*Y sequences, f(ins) varies by as much as 3 orders of magnitude, being smallest with G flanking the lesions on the 3'-side and highest with G flanking the adducts on the 5'-side. One-step primer extension efficiencies just beyond the lesions (f(ext)) are generally smaller than f(ins) and also depend on base sequence. However, reasonably efficient translesion bypass of the (+)-trans-[BP]-N(2)-dG adducts is observed in all sequences in running-start experiments with full, or nearly full, primer extension being observed under conditions of [dNTP] > K(m). The key features here are the relatively robust values of the kinetic parameters V(max) that are either diminished to a moderate extent or even enhanced in the presence of the (+)-trans-[BP]-N(2)-dG adducts. In contrast to the small effects of the lesions on V(max), the apparent K(m) values are orders of magnitude greater in XG*Y than in the unmodified XGY sequences. Thus the bypass of (+)-trans-[BP]-N(2)-dG adducts under conditions when [dNTP] < K(m) is quite inefficient. These considerations may be of importance in vivo where [dNTP] <or= K(m), and the translesion bypass of the (+)-trans-[BP]-N(2)-dG by pol kappa may be significantly less efficient than in vitro at higher dNTP concentrations. The base sequence-dependent features of translesion bypass are discussed in terms of the possible conformations of the adducts and the known structural features of bypass polymerases.
Huang
Xuanwei
X
Department of Chemistry, New York University, 31 Washington Place, New York, New York 10003-5180, USA.
Kolbanovskiy
Alexander
A
Wu
Xiaohua
X
Zhang
Yanbin
Y
Wang
Zhigang
Z
Zhuang
Ping
P
Amin
Shantu
S
Geacintov
Nicholas E
NE
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000138:chemical synthesis* / Q000737:chemistry; D000227:Adenine Nucleotides / Q000737:chemistry; D001483:Base Sequence; D002384:Catalysis; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry*; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D003845:Deoxycytosine Nucleotides / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D009153:Mutagens / Q000138:chemical synthesis* / Q000737:chemistry; D009687:Nuclear Proteins / Q000737:chemistry*; D013698:Templates, Genetic; D000099270:Y-Family DNA Polymerases
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D000227:Adenine Nucleotides; D002868:Chromosomal Proteins, Non-Histone; D018736:DNA Adducts; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D009153:Mutagens; D009687:Nuclear Proteins; C024107:2'-deoxycytidine 5'-triphosphate; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases; C118146:TENT4A protein, human