Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding.
We characterized nine helicase-deficient mutants of bacteriophage T7 helicase-primase protein (4A') prepared by random mutagenesis as reported in the accompanying paper (Rosenberg, A. H., Griffin, K., Washington, M. T., Patel, S. S., and Studier, F. W. (1996) J. Biol. Chem. 271, 26819-26824). Mutants were selected from each of the helicase-conserved motifs for detailed analysis to understand better their function. In agreement with the in vivo results, the mutants were defective in helicase activity but were active in primase function. dTTP hydrolysis, DNA binding, and hexamer formation were examined. Three classes of defective mutants were observed. Group A mutants (E348K, D424N, and S496F), defective in dTTP hydrolysis, lie in motifs 1a, 2, and 4 and are possibly involved in NTP binding/hydrolysis. Group B mutants (R487C and G488D), defective in DNA binding, lie in motif 4 and are responsible directly or indirectly for DNA binding. Group C mutants (G116D, A257T, S345F, and G451E) were not defective in any of the activities except the helicase function. These mutants, scattered throughout the protein, appear defective in coupling dTTPase activity to helicase function. Secondary structural predictions of 4A' and DnaB helicases resemble the known structures of RecA and F1-ATPase enzymes. Alignment shows a striking correlation in the positions of the amino acids that interact with NTP and DNA.
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
M T
MT
Washington
A H
AH
Rosenberg
K
K
Griffin
F W
FW
Studier
S S
SS
Patel
D000595:Amino Acid Sequence; D001704:Biopolymers; D019915:DNA Primase; D004261:DNA Replication; D004274:DNA, Recombinant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000378:metabolism*
D001704:Biopolymers; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; C024157:thymidine 5'-triphosphate