Purification of the DNA binding domain of herpes simplex virus type 1 immediate-early protein Vmw175 as a homodimer and extensive mutagenesis of its DNA recognition site

Everett, R.D., Elliott, M., Hope, G. and Orr, A. (1991) Purification of the DNA binding domain of herpes simplex virus type 1 immediate-early protein Vmw175 as a homodimer and extensive mutagenesis of its DNA recognition site. Nucleic Acids Research, 19(18), pp. 4901-4908. (doi: 10.1093/nar/19.18.4901)

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Abstract

The herpes simplex virus type 1 (HSV-1) Immediate- Early (IE) polypeptide Vmw175 Is essential for the activation of transcription from viral early and late promoters. Vmw175 also reduces the activity of its own (IE-3) promoter In transfectlon assays. Both transactivatlon and repression mediated by Vmw175 require the integrity of a conserved domain of the polypeptide which has been shown to bind to specific DNA sequences. We have investigated the DNA sequence requirements for Vmw175 binding using a randomly mutated target. The results indicate that the binding site covers a region of 13 nucleotldes divided into proximal and distal parts which are consistent with the consensus ATCGTNNNNNYSG. We have also expressed several different constructs encompassing the DNA binding domain of Vmw175 in bacteria, and obtained preparations of greater than 90% purity. The DNA binding domain is a dimer in solution, and binds DNA with a specificity similar to that of the Intact protein, although the smallest DNA binding competent protein has a slightly reduced specificity.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Everett, Professor Roger and Orr, Mrs Anne and Hope, Dr Graham
Authors: Everett, R.D., Elliott, M., Hope, G., and Orr, A.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Nucleic Acids Research
Publisher:Oxford University Press
ISSN:0305-1048
ISSN (Online):1362-4962
Copyright Holders:Copyright © 1991 Oxford University Press
First Published:First published in Nucleic Acids Research 19(18):4901-4908
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher

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