Mapping interactions between the catalytic domain of resolvase and its DNA substrate using cysteine-coupled EDTA-iron

Abstract

Single cysteine-substituted mutants of gamma delta resolvase have been covalently modified using a novel sulfhydryl-specific EDTA derivative, EDTA-2-aminoethyl 2-pyridyl disulfide (EPD). Iron, chelated by the coupled EDTA and in the presence of reducing agent, generates reactive oxygen species that result in localized cleavage of the DNA to which resolvase is bound. The procedure provides valuable information on two fronts. First, it allows the identification of regions or surfaces of the protein that are in close proximity to DNA even though they may not be part of the DNA-binding domain. Second, it allows identification of the portions of DNA that are closest to each EDTA-derivatized cysteine, since the DNA cleavages observed are highly localized and their efficiency drops rapidly as a function of the distance between the EDTA-Fe complex and the deoxyribose target. We have used the procedure to investigate the interaction of gamma delta resolvase with the three DNA binding sites that constitute its recombination substrate, res. The data indicate that the two N-terminal domains of a resolvase dimer interact symmetrically with site I, which contains the recombination cross-over point, but asymmetrically with the accessory sites, II and III. The patterns of DNA cleavage obtained with several different EDTA-coupled mutants have enabled us to propose a model for the interaction between resolvase and site I.

Site-specific HU binding in the Mu transpososome: conversion of a sequence-independent DNA-binding protein into a chemical nuclease.

“…H~) 2-S-S-HU-n uc I ease EPD. Fe (I I I) Mazzarelli et al 1993). Because HId is devoid of cysteines, 2-iminothiolane (Traut's reagent) was used to add sulfhydryl groups at primary amines (Traut et al 1973;Jue et al 1978).…”

“…This approach presents several advantages. The iron-EDTA complex can be stably introduced into the native protein (Ermficora et al 1992} and does not promote spontaneous cleavage of the DNA (Ebright et al 1992;Mazzarelli et al 1993). This permits assembly of higher order nucleoprotein structures that require supercoiled substrates.…”

“…This permits assembly of higher order nucleoprotein structures that require supercoiled substrates. Upon the addition of a reducing agent, DNA cleavage ensues rapidly, is highly localized, and shows little or no sequence specificity (Ebright et al 1992;Mazzarelli et al 1993).…”

“…Fe{II)] reagent has been used to cleave DNA within -30 A of the protein derivatization site {Ebright et al 1992; Mazzarelli et al 1993). It reacts with sulfhydryl residues and becomes coupled to proteins through a disulfide bond (Ebright et al 1992;Ermficora et al 1992; HU protein 2-iminothiolane thiolated HU …”

Determination of the structure of the DNA binding domain of γΔ resolvase in solution

“…The resolvase DBD protects against nuclease digestion at adjacent major and minor grooves of the cognate DNA sequence, and the isolated resolvase DBD interacts at the res subsites similarly to the intact resolvase (Abdel-Meguid et al, 1984;Rimphanitchayakit et al, 1989). The catalytic domain of the intact protein provides additional contacts on the opposite face of the DNA duplex but does not interact with DNA independently (Abdel-Meguid et al, 1984;Falvey & Grindley, 1987;Mazzarelli et al, 1993). The cooperative interaction of resolvase with res results in bending at each of the 3 res binding sites and looping of the DNA between res sites I and I1 .…”