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PDBsum entry 2w7o
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DNA binding protein
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PDB id
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2w7o
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References listed in PDB file
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Key reference
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Title
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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.
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Authors
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A.Irimia,
R.L.Eoff,
F.P.Guengerich,
M.Egli.
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Ref.
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J Biol Chem, 2009,
284,
22467-22480.
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PubMed id
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Abstract
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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.
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