Altered order of substrate binding by DNA polymerase X from African swine fever virus

Sandeep Kumar, Marina Bakhtina, Ming Daw Tsai

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

A sequential ordered substrate binding established previously for several DNA polymerases is generally extended to all DNA polymerases, and the characterization of novel polymerases is often based on the assumption that the enzymes should productively bind DNA substrate first, followed by template-directed dNTP binding. The comprehensive kinetic study of DNA polymerase X (Pol X) from African swine fever virus reported here is the first analysis of the substrate binding order performed for a low-fidelity DNA polymerase. A classical steady-state kinetic approach using substrate analogue inhibition assays demonstrates that Pol X does not follow the bi-bi ordered mechanism established for other DNA polymerases. Further, using isotope-trapping experiments and stopped-flow fluorescence assays, we show that Pol X can bind Mg2+·dNTPs in a productive manner in the absence of DNA substrate. We also show that DNA binding to Pol X, although rapid, may not always be productive. Furthermore, we show that binding of Mg 2+·dNTP to Pol X facilitates subsequent formation of the catalytically competent Pol X·DNA·dNTP ternary complex, whereas DNA binding prior to dNTP binding brings the enzyme into a nonproductive conformation where subsequent nucleotide substrate binding is hindered. Together, our results suggest that Pol X prefers an ordered sequential mechanism with Mg2+·dNTP as the first substrate.

Original languageEnglish
Pages (from-to)7875-7887
Number of pages13
JournalBiochemistry
Volume47
Issue number30
DOIs
StatePublished - 29 Jul 2008
Externally publishedYes

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