ROLE OF CYSTEINE62 IN DNA RECOGNITION BY THE P50 SUBUNIT OF NF-ALEPH-B

A powerful chemical modification procedure has been developed to defin e determinants of DNA recognition by the p50 subunit of NF-kappaB. Dif ferential labelling with [C-14] iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition sit e 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 a nalysis indicated that the C62S mutant bound to its DNA recognition si te with a 10-fold larger dissociation constant than the wild type prot ein, while the other two mutants bound with an intermediate affinity. Dissociation rate constant measurements correlated well with the disso ciation constants for the wild type, C119S, and C273S p50 proteins, wh ereas the p50 C62S-DNA complex dissociated anomalously quickly. Compet ition analyses with oligonucleotide variants of the DNA recognition si te 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 sulphy dryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappaB.