title: Phenylalanine 171 is a molecular brake for translesion synthesis across benzo[a]pyrene-guanine adducts by human DNA polymerase kappa.
abstract: Human cells possess multiple specialized DNA polymerases (Pols) that bypass a variety of DNA lesions which otherwise would block chromosome replication. Human polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. To better understand the relationship between the structural features in the active site and lesion bypass by Pol κ, we mutated codons corresponding to amino acids appearing close to the adducts in the active site, and compared bypass efficiencies. Remarkably, the substitution of alanine for phenylalanine 171 (F171), an amino acid conserved between Pol κ and its bacterial counterpart Escherichia coli DinB, enhanced the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG 18-fold. This substitution affected neither the fidelity of TLS nor the efficiency of dCMP incorporation opposite normal guanine. This amino acid change also enhanced the binding affinity of Pol κ to template/primer DNA containing (-)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 functions as a molecular brake for TLS across BPDE-N(2)-dG by Pol κ and that the F171A derivative of Pol κ bypasses these DNA lesions more actively than does the wild-type enzyme.
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Sassa
authors.forename: Naoko
authors.initials: N
authors.lastname: Niimi
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Fujimoto
authors.forename: Atsushi
authors.initials: A
authors.lastname: Katafuchi
authors.forename: Petr
authors.initials: P
authors.lastname: Grúz
authors.forename: Manabu
authors.initials: M
authors.lastname: Yasui
authors.forename: Ramesh C
authors.initials: RC
authors.lastname: Gupta
authors.forename: Francis
authors.initials: F
authors.lastname: Johnson
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Ohta
authors.forename: Takehiko
authors.initials: T
authors.lastname: Nohmi
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D001483:Base Sequence; D001564:Benzo(a)pyrene / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000099270:Y-Family DNA Polymerases
chemical_list: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D001564:Benzo(a)pyrene; D018736:DNA Adducts; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D029968:Escherichia coli Proteins; D010649:Phenylalanine; D011994:Recombinant Proteins; C094399:DinB protein, E coli; C418058:benzo(a)pyrene 7,8-diol-9,10-epoxide-N2-deoxyguanosine; C056612:benzo(a)pyrene-DNA adduct; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases