Altered order of substrate binding by DNA polymerase X from African Swine Fever virus.
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 Mg (2+).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 Mg (2+).dNTP as the first substrate.
Kumar
Sandeep
S
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Bakhtina
Marina
M
Tsai
Ming-Daw
MD
D000358:African Swine Fever Virus / Q000201:enzymology*; D000818:Animals; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003854:Deoxyribonucleotides; C024107:2'-deoxycytidine 5'-triphosphate; C109534:DNA polymerase X; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; C026600:2'-deoxyadenosine triphosphate