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Abstract
The human DNA polymerase kappa homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces "-1" frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N(2)-etheno-2'-deoxyguanosine, with a larger stacking surface than canonical purines, showed the highest frequency of formation of -1 frameshift deletions. Dpo4 T239W, a mutant we had previously shown to produce fluorescence changes attributed to conformational change following dNTP binding opposite cognate bases (Beckman, J. W., Wang, Q., and Guengerich, F. P. (2008) J. Biol. Chem. 283, 36711-36723), reported similar conformational changes when the incoming dNTP complemented the base following a templating purine base or bulky adduct (i.e. the "+1" base). However, in all mispairing cases, phosphodiester bond formation was inefficient. The frequency of -1 frameshift events and the associated conformational changes were not dependent on the context of the remainder of the sequence. Collectively, our results support a mechanism for -1 frameshift deletions by Dpo4 that involves formation of active complexes via a favorable conformational change that skips the templating base, without causing slippage or flipping out of the base, to incorporate a complementary residue opposite the +1 base, in a mechanism previously termed "dNTP-stabilized incorporation." The driving force is attributed to be the stacking potential between the templating base and the incoming dNTP base.
Highlights
We observed rapid fluorescence increases that corresponded to noncomplementary dNTP binding but without fluorescence decreases. We investigated these events associated with Ϫ1 frameshifts by examining the fluorescence changes of Dpo4 T239W and comparing the data with quantitative analyses of product formation using steady-state kinetics and LC/MS-MS
Shift because of the inability to proceed with normal base pairing at the insertion site (Fig. 1) (if all four dNTPs are present, the primer is prone to rapid incorporation and extension in an accurate mode, not revealing potential products resulting from slower misincorporations and frameshifts [29, 31, 32])
Translesion DNA polymerases appear to be prone to frameshift mutations (9 –11, 19). The reason for this behavior may be that these polymerases form relatively few protein contacts with the incoming dNTP and primer-template complex to help anchor the substrates at one site, leading to formation of alternative active site configurations that lead to different products
Summary
X indicates A, C, G, or T; Xdd indicates a terminal 2Ј,3Ј-dideoxy-X (incapable of phosphodiester bond formation); YYY indicates GGC, GGT, AAC, CAG, GAG, GAC, TTC, GTC, TCG, CCT, GCT, GCA, G(O6-MeG)C, G(8-oxoG)C, G(1,N2-⑀G)C, G(1,N2-⑀-G)T, or G(AP)C. Dpo T239W produced fluorescence changes upon binding of a complementary dNTP, a rapid increase in fluorescence upon formation of an active ternary complex followed by a slower decrease following phosphodiester bond formation, with the latter attributed to relaxation of the conformation. We observed rapid fluorescence increases that corresponded to noncomplementary dNTP binding but without fluorescence decreases (i.e. phosphodiester bond formation was relatively inefficient). We investigated these events associated with Ϫ1 frameshifts (because they only occurred when the incoming dNTP complemented the ϩ1 base) by examining the fluorescence changes of Dpo T239W and comparing the data with quantitative analyses of product formation using steady-state kinetics and LC/MS-MS. We observed that formation of Ϫ1 frameshift products was facilitated by the presence of a templating purine or an adduct with relatively high stacking potential (1,N2-⑀-G), and we conclude that such events involve a rapid and favorable conformational change despite very slow phosphodiester bond formation
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