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Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding
Journal article   Open access   Peer reviewed

Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding

M Washington, A H Rosenberg, K Griffin, F Studier and S Patel
The Journal of biological chemistry, Vol.271(43), pp.26825-26834
01/01/1996
DOI: 10.1074/jbc.271.43.26825
PMID: 8900164
url
https://doi.org/10.1074/jbc.271.43.26825View
Published (Version of record) Open Access

Abstract

We characterized nine helicase-deficient mutants of bacteriophage T7 helicase-primase protein (4A') prepared by random mutagenesis. 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 F sub(1)-ATPase enzymes. Alignment shows a striking correlation in the positions of the amino acids that interact with NTP and DNA.
 phage T7

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