A DNA polymerase with specificity for five base pairs.

The X family DNA polymerase from African swine fever virus (Pol X) has recently been characterized as the smallest known nucleotidyl transferase and has been suggested to play a role in DNA repair analogous to that of its mammalian sequence homologue, DNA polymerase â (Pol â).1 In this study an indepth kinetic analysis of Pol X, including catalytic efficiency and fidelity measurements for all possible base pairs, demonstrates that Pol X is the least faithful, or most error-prone, of all polymerases studied to date, with a specific preference for five base pairs including the four Watson-Crick base pairs plus one mismatched pair. Our conclusion that Pol X is the least faithful polymerase is based on pre-steady-state kinetics, using model DNA substrates (Figure 1). We have first measured single turnover (with enzyme in excess of DNA substrate) saturation kinetics for all 16 possible base pairs in single-gapped DNA substrates. It has previously been shown that Pol X is a processive enzyme only when acting on gapped substrate,1 and our observation that burst kinetics are observed only with gapped DNA (data not shown) confirm this and suggest that gapped DNA is likely to be the enzyme’s natural substratesas is the case for Pol â. Single turnover experiments allow direct determination of the principal kinetic parameters kpol (the pseudo-first-order catalytic rate constant) and Kd,app (the apparent equilibrium constant for dissociation of nucleotide triphosphate from the enzyme‚DNA complex) of nucleotide incorporation. The ratio kpol/Kd is the definition of substrate specificity (it is also termed “catalytic efficiency”), and thus comparison of this value for correct and incorrect incorporations gives a quantitative measurement of the fidelity for a polymerase. The results shown in Table 1 indicate an activity which is incompatible with a repair function. The enzyme has relatively low catalytic efficiency, on average 1/5000th that of Pol â (an enzyme known to function in base excision repair, or BER2) for correct base-pair incorporations.3 More strikingly, Pol X has exceptionally low fidelities, ranging from 7700 for the C:C base pair to 1.9 for the G:G base pair. As the fidelitysdefined as [(kpol/Kd,app)cor + (kpol/Kd,app)inc]/(kpol/Kd,app)inc where the subscripts “cor” and “inc” refer to the correct and incorrect incorporation, respectivelysis the inverse of the error frequency, this indicates that the enzyme has no substrate specificity for a correct base pair (G:C) relative to the corresponding incorrect base pair (G:G). While the entire fidelity spectrum for Pol X is remarkably low, this absence of discrimination between the G:C and G:G base pairs represents the lowest nucleotide incorporation specificity ever observed for a templatedirected nucleotide polymerase. Human Pol η, an enzyme recently determined to be the most error-prone polymerase,4 is at least 10 times more faithful than Pol X in this instance. As illustrated in Figure 2, Pol X appears to catalyze formation of five base pairs (the four Watson-Crick pairs plus G:G) with comparable efficiency, selecting against the other 11 base pairs with fidelities ranging from modest to very low. Another important characteristic illustrated in Figure 2 is that, while the catalytic efficiency of Pol X is generally suppressed relative to that of Pol â, formation of the G:G mismatch is enhanced by nearly 8-fold. Thus, the mutagenicity of Pol X is the product of an impaired ability to form correct base pairs coupled with an enhanced ability to form the select mispair. † Department of Chemistry. ‡ Department of Biochemistry. § The Ohio State Biochemistry Program. (1) Oliveros, M.; Yáñez, R. J.; Salas, M. L.; Salas, J.; Viñuela, E.; Blanco, L. J. Biol. Chem. 1997, 272, 30899-30910. (2) Sobol, R. W.; Horton, J. K.; Kuhn, R.; Gu, H.; Singhal, R. K.; Prasad, R.; Rajewsky, K.; Wilson, S. H. Nature 1996, 379, 183-186. (3) Ahn, J.; Kraynov, V. S.; Zhong, X.; Werneburg, B. G.; Tsai, M.-D. Biochem. J. 1998, 331, 79-87. (4) Matsuda, T.; Bebenek, K.; Masutani, C.; Hanaoka, F.; Kunkel, T. A. Nature 2000, 404, 1011-1013. Figure 1. Schematic representation of gapped (A) and nongapped (B) DNA substrate. The difference between the two is the presence or absence of a downstream oligonucleotide, which is phosphorylated at the 5′-terminus.