Sequence and functional-group specificity for cleavage of DNA junctions byRuvC of Escherichia coli

RuvC is the DNA junction-resolving enzyme of Escherichia coli. While the en zyme binds to DNA junctions independently of base sequence, it exhibits con siderable sequence selectivity for the phosphodiester cleavage reaction. We have analyzed the sequence specificity using a panel of DNA junctions, mea suring the rate of cleavage of each under single-turnover conditions. We ha ve found that the optimal sequence for cleavage can be described by (A simi lar to T)TT down arrow(C > G-A), where down arrow denotes the position of b ackbone scission. Cleavage is fastest when the cleaved phosphodiester linka ge is located at the point of strand exchange. However, cleavage is possibl e one nucleotide 3' of this position when directed by the sequence, with a rate that is 1 order of magnitude slower than the optimal. The maximum sequ ence discrimination occurs at the central TT in the tetranucleotide site, w here any alteration of sequence results in a rate reduction of at least 100 -fold and cleavage is undetectable for some changes, However, certain seque nces in the outer nucleotides are strongly inhibitory to cleavage. Introduc tion of base analogues around the cleavage site reveals a number of importa nt functional groups and suggests that major-groove contacts in the center of the tetranucleotide are important for the cleavage process. Since RuvC b inds to all the variant junctions with very similar affinity, any contacts affecting the rate of cleavage must be primarily important in the transitio n state. Introduction of the optimal cleavage sequence into a three-way DNA junction led to relatively efficient cleavage by RuvC, at a rate only 3-fo ld slower than the optimal four-way junction. This is consistent with a pro tein-induced alteration in the conformation of the DNA.