The Structure of Bacillus subtilis RecU Holliday Junction Resolvase and Its Role in Substrate Selection and Sequence-Specific Cleavage

Abstract

We have determined the structure of the enzyme RecU from Bacillus subtilis, that is the general Holliday junction resolving enzyme in Gram-positive bacteria. The enzyme fold reveals a striking similarity to a class of resolvase enzymes found in archaeal sources and members of the type II restriction endonuclease family to which they are related. The structure confirms the presence of active sites formed around clusters of acidic residues that we have also shown to bind divalent cations. Mutagenesis data presented here support the key role of certain residues. The RecU structure suggests a basis for Holliday junction selectivity and suggests how sequence-specific cleavage might be achieved. Models for a resolvase-DNA complex address how the enzyme might organize junctions into an approximately 4-fold symmetric form.

Interaction of Branch Migration Translocases with the Holliday Junction-resolving Enzyme and Their Implications in Holliday Junction Resolution

“…4B). We hypothesized that (i) RecU transiently positions RuvB⅐ATP␥S at the center of the HJ, and (ii) RuvB⅐ATP␥S may promote the distortion of HJ DNA, and by this way it facilitates HJ resolution (23,31). This is in agreement with the observation that ternary complexes are more stable in the presence of ATP␥S.…”

“…The radiolabeled HJ-J1 and HJ-J3 were assembled and purified from four oligonucleotides each of 80 nucleotides as previously described (23,38). The radiolabeled HJ-jbm6 was assembled from four 40-nucleotide oligonucleotides and gel-purified.…”

“…Proteins-RecU, RecUE36A, RecUR31A, RecUY80A, and RuvB were purified, and their concentrations were determined as previously described (23,29,30,36,37). Cells overexpressing the RuvA protein were harvested and resuspended in buffer A (50 mM Tris-HCl, pH 8.5, 1 mM EDTA, 10% glycerol) containing 100 mM KCl and lysed by sonication.…”

“…Based on the RecU structure, we proposed a model of how RecU binds to the HJ DNA. The RecU stalk region, by penetrating in the center of the HJ, distorts it so that the HJ adopts a square planar conformation with a central hole (23). This model was recently confirmed by the identification of residues located in the stalk region that are essential for HJ recognition and flexibility (30,31).…”

“…RecU) (6). The RecU HJ resolvase is a dimeric enzyme that shares structural similarity with viral (T7 Endo I) and archaeal (Hje and Hjc) resolvases (23). From the results obtained with E. coli enzymes, it is believed that bacterial HJ resolvases work in concert with RuvAB.…”