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PMID: 8493089 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Role of cysteine62 in DNA recognition by the P50 subunit of NF-kappa B.

Nucleic acids research ·Vol. 21 ·No. 8 ·1993-04-25 ·Pages 1727-34

Matthews JR, Kaszubska W, Turcatti G, Wells TN, Hay RT

Abstract

A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunit of NF-kappa B. Differential labelling with [14C] iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition site of the protein. To determine the importance of this cysteine residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined. Scatchard analysis indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an intermediate affinity. Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly. Competition analyses with oligonucleotide variants of the DNA recognition site and nonspecific E. coli DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was impaired in its ability to discriminate between specific and non-specific DNA. Thus the sulphydryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappa B.

MeSH Terms
Amino Acid Sequence Base Sequence Binding Sites Cloning, Molecular Cysteine/metabolism DNA, Bacterial/metabolism Escherichia coli Kinetics Molecular Sequence Data Mutation NF-kappa B/chemistry,genetics,metabolism Oxidation-Reduction
Chemicals
DNA, Bacterial NF-kappa B Cysteine
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Matthews J R
School of Biological and Medical Sciences, University of St Andrews, Fife, UK.
Kaszubska W
Turcatti G
Wells T N
Hay R T
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/21.8.1727
Published
1993-04-25
Pages
1727-34
Language
English
Region
England
NLM ID
0411011
PMCID
PMC309407
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/21.8.1727 PMC Full Text View journal details
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