Structural and functional elucidation of the mechanism promoting error-prone synthesis by human DNA polymerase kappa opposite the 7,8-dihydro-8-oxo-2'-deoxyguanosine adduct.
Human polymerase kappa (hPol kappa) is one of four eukaryotic Y-class DNA polymerases and may be an important element in the cellular response to polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which can lead to reactive oxygenated metabolite-mediated oxidative stress. Here, we present a detailed analysis of the activity and specificity of hPol kappa bypass opposite the major oxidative adduct 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG). Unlike its archaeal homolog Dpo4, hPol kappa bypasses this lesion in an error-prone fashion by inserting mainly dATP. Analysis of transient-state kinetics shows diminished "bursts" for dATP:8-oxoG and dCTP:8-oxoG incorporation, indicative of non-productive complex formation, but dATP:8-oxoG insertion events that do occur are 2-fold more efficient than dCTP:G insertion events. Crystal structures of ternary hPol kappa complexes with adducted template-primer DNA reveal non-productive (dGTP and dATP) alignments of incoming nucleotide and 8-oxoG. Structural limitations placed upon the hPol kappa by interactions between the N-clasp and finger domains combined with stabilization of the syn-oriented template 8-oxoG through the side chain of Met-135 both appear to contribute to error-prone bypass. Mutating Leu-508 in the little finger domain of hPol kappa to lysine modulates the insertion opposite 8-oxoG toward more accurate bypass, similar to previous findings with Dpo4. Our structural and activity data provide insight into important mechanistic aspects of error-prone bypass of 8-oxoG by hPol kappa compared with accurate and efficient bypass of the lesion by Dpo4 and polymerase eta.
Irimia
Adriana
A
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Eoff
Robert L
RL
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Guengerich
F Peter
FP
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Egli
Martin
M
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
D000080242:8-Hydroxy-2'-Deoxyguanosine; D001483:Base Sequence; D002384:Catalysis; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D016944:Sulfolobus; D014961:X-Ray Diffraction; D000099270:Y-Family DNA Polymerases
D000080242:8-Hydroxy-2'-Deoxyguanosine; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D005656:Fungal Proteins; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases