Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[a]pyrene dG adduct.
DNA polymerase κ (Polκ) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide (BPDE)-N(2)-deoxyguanine adducts efficiently and accurately. The unique features of Polκ, a large structure gap between the catalytic core and little finger domain and a 90-residue addition at the N terminus known as the N-clasp, may give rise to its special translesion capability. We designed and constructed two mouse Polκ variants, which have reduced gap size on both sides [Polκ Gap Mutant (PGM) 1] or one side flanking the template base (PGM2). These Polκ variants are nearly as efficient as WT in normal DNA synthesis, albeit with reduced accuracy. However, PGM1 is strongly blocked by the 10S (+)-trans-anti-BPDE-N(2)-dG lesion. Steady-state kinetic measurements reveal a significant reduction in efficiency of dCTP incorporation opposite the lesion by PGM1 and a moderate reduction by PGM2. Consistently, Polκ-deficient cells stably complemented with PGM1 GFP-Polκ remained hypersensitive to BPDE treatment, and complementation with WT or PGM2 GFP-Polκ restored BPDE resistance. Furthermore, deletion of the first 51 residues of the N-clasp in mouse Polκ (mPolκ(52-516)) leads to reduced polymerization activity, and the mutant PGM2(52-516) but not PGM1(52-516) can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPolκ(52-516) retains BPDE bypass activity. We conclude that the structural gap physically accommodates the bulky aromatic adduct and the N-clasp is essential for the structural integrity and flexibility of Polκ during translesion synthesis.
Laboratory of Cancer Genomics and Individualized Medicine, Beijing Institute of Genomics and State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Yang
Y
Liu
Yeran
Y
Yang
Tie-Shan
TS
Tang
Hui
H
Zhang
Zhifeng
Z
Wang
Errol
E
Friedberg
Wei
W
Yang
Caixia
C
Guo
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000494:pharmacology; D000818:Animals; D001564:Benzo(a)pyrene / Q000737:chemistry / Q000378:metabolism; D001580:Benzopyrenes / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D017931:DNA Primers / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004622:Embryo, Mammalian / Q000473:pathology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D001564:Benzo(a)pyrene; D001580:Benzopyrenes; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D050505:Mutant Proteins; C478198:benzo(a)pyrene N2-dG adduct; C498613:Polk protein, mouse; D000099270:Y-Family DNA Polymerases
polycyclic aromatic hydrocarbons; translesion DNA synthesis