IDENTIFICATION AND CHARACTERIZATION OF THE N-ETHYLMALEIMIDE-SENSITIVESITE IN LAMBDA-INTEGRASE

Integrase (Int) of bacteriophage lambda is a heterobivalent DNA bindin g protein and a type I topoisomerase. Upon modification with N-ethylma leimide (NEM), a sulfhydryl-directed reagent, Int loses its capacity t o bind ''arm-type'' DNA sequences and, consequently, to carry out reco mbination; however, its ability to bind ''core-type'' sequences and it s topoisomerase activity are unaffected, In this report, the NEM-sensi tive site was identified by modifying Int with [C-14]NEM. Following cl eavage by formic acid, which cleaves Asp-Pro bonds, and fractionation on a Fractogel HW-50 (F) sizing column, the fragment containing the pr imary site of [C-14]NEM incorporation was subjected to amino acid sequ encing. The results indicate that the primary site of [C-14]NEM incorp oration is in the peptide-spanning amino acid residues 1-28, which con tains a cysteine at position 25. To confirm that Cys-25 is the target of NEM reactivity, site-directed mutagenesis was used to change this c ysteine to alanine or serine. The mutant protein is not chemically mod ified by NEM and shows no loss of activity after NEM treatment. The fa ct that C25A and C25S both retain full recombination activity indicate s that the SH group of Cys-25 does not provide any critical contacts, either with arm-type DNA or with other parts of the Int protein to for m the arm-type recognition pocket. The loss of arm-type DNA binding an d the con-comitant loss of recombination function as a result of NEM m odification must be due to the presence of the maleimide moiety and no t due to loss of a critical cysteine contact.