Analysis of Substrate Specificity of the RuvC Holliday Junction Resolvase with Synthetic Holliday Junctions

Abstract

The Escherichia coli RuvC protein endonucleolytically resolves Holliday junctions, which are formed as intermediates during genetic recombination and recombination repair. Previous studies using model Holliday junctions suggested that a certain size of central core of homology and a specific sequence in the junction were required for efficient cleavage by RuvC, although not for binding. To determine the minimum length of sequence homology required for RuvC cleavage, we made a series of synthetic Holliday junctions with various lengths of homologous sequence in the core region. It was demonstrated that a monomobile junction possessing only 2 base pairs of the homology core was efficiently cleaved by RuvC. To study the sequence specificity for cleavage, we made 16 bimobile junctions, which differed only in the homologous core sequence. Among them, 6 junctions were efficiently cleaved. Cleavage occurred by introduction of nicks symmetrically at the 3'-side of thymine in all cases. However, the nucleotide bases at the 3'-side of the thymines were not always identical between the two strands nicked. These results suggest that RuvC recognizes mainly topological symmetry of the Holliday junction but not the sequence symmetry per se, that the thymine residue at the cleavage site plays an important role for RuvC-mediated resolution, and that a long homologous core sequence is not essential for cleavage.

Structure and Metal Binding Properties of a Poxvirus Resolvase

“…In the CPV resolvase model, the ␣2 helix is shorter, and the enzyme can only contact the sugar- phosphate backbone at the junction center. These observations are consistent with and may explain the relative lack of sequence specificity near the junction branch point for the poxvirus enzymes (12,13), compared with the strong preferences observed for E. coli RuvC (43,44).…”

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

“…We have suggested that RuvC binding involves two modes of DNA binding: structure specific binding to recognize the configuration of the Holliday junction and catalytically competent interactions (Shida et al 1996). It has also been suggested that RuvC binding to the junction induces a junction distortion and disrupts several base pairs at the crossover point (Bennett et al 1993;Bennett & West 1995a).…”

“…Various biochemical approaches using model Holliday junctions have been applied to elucidate the reaction mechanism of the RuvC-mediated resolution. RuvC cleaves the junctions by introducing two symmetrically related nicks in the continuous strands near the crossover point (Dunderdale et al 1991;Iwasaki et al 1991;Bennett & West 1995bShida et al 1996). RuvC preferentially cleaves junctions with certain core DNA sequences containing A:T base pairs at the 3 0 side of T (Shah et al 1994;Shida et al 1996).…”

“…Theses results indicate that binding to, and cleavage of, the Holliday junction are distinct steps in junction resolution. In addition, the binding of RuvC to the junction has been suggested to involve two modes of DNA binding: structure specific binding to recognize the configuration of the Holliday junction, and catalytically competent interactions leading to cleavage (Shida et al 1996). The latter is likely to involve specific interactions between particular residues of RuvC and specific nucleotides of the junction DNA.…”

Evidence that Phenylalanine 69 in Escherichia coliRuvC Resolvase Forms a Stacking Interaction during Binding and Destabilization of a Holliday Junction DNA Substrate

“…This is likely to be an important characteristic of the interaction between RuvC and its DNA substrate (14,16). (iii) In the presence of metal ions such as Mg 2ϩ and Mn 2ϩ , RuvC resolves the junctions by introducing symmetrical nicks in the continuous (noncrossover) strands (14,15,17), and (iv) cleavage occurs preferentially at the consensus sequence 5Ј-(A/ T)TT2(G/C), where 2 represents a cleavage site close to the crossover point (18,19). Following cleavage, the resolution process is completed by DNA ligase, which rejoins the 5Ј-P and 3Ј-OH termini in the nicked duplex products (13,14).…”