Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation.
A rapid purification procedure produces milligram amounts of the T7 gene 4A' primase/helicase, 4B helicase, and the wild-type 4AB proteins expressed from the clones described in the accompanying paper (Rosenberg, A. H., Patel, S. S., Johnson, K. A., and Studier, F. W. (1992) J. Biol. Chem. 267, 15005-15012). Purified 4A' protein (in which the wild-type methionine at amino acid 64 has been replaced by leucine to eliminate the 4B initiation codon) appears to be equivalent to the wild-type 4A protein in primase, helicase, and NTPase activities. Gel filtration chromatography and polyacrylamide gel electrophoresis of native proteins indicate that the 4A' and 4B proteins form homodimers and heterodimers in solution. Heterodimer formation presumably accounts for an observed 3-fold increase in the primase activity of 4A' upon addition of 4B that lacks primase activity of its own. Steady-state k(cat) and Km values for hydrolysis of the nucleoside triphosphates ATP, dATP, dTTP, and dGTP were measured for 4A', 4B, 4A'B (1:1), and wild-type 4AB (1:2) proteins. The dependence of the dNTPase activities on the concentration was hyperbolic, suggesting single or noncooperative binding sites, whereas ATPase activity was sigmoidal, suggesting more than one ATP binding site. The k(cat)/Km ratios for hydrolysis of the dNTPs by the four protein preparations were within a factor of 6 of each other. The 1:1 mixture of 4A'B had the highest k(cat)/Km ratios, with a preference for dATP and dTTP.
Patel
S S
SS
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Rosenberg
A H
AH
Studier
F W
FW
Johnson
K A
KA
D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D019915:DNA Primase; D004279:DNA, Viral / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005814:Genes, Viral; D006868:Hydrolysis; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism
D004279:DNA, Viral; D007527:Isoenzymes; D014764:Viral Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases