Abstract
Holliday junction (HJ) resolvases are structure-specific endonucleases that cleave four-way DNA junctions (HJs) generated during DNA recombination and repair. Bacterial RuvC, a prototypical HJ resolvase, functions as homodimer and nicks DNA strands precisely across the junction point. To gain insights into the mechanisms underlying symmetrical strand cleavages by RuvC, we performed crystallographic and biochemical analyses of RuvC from Thermus thermophilus (T.th. RuvC). The crystal structure of T.th. RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but T.th. RuvC has a more tightly associated dimer interface possibly reflecting its thermostability. The binding mode of a HJ-DNA substrate can be inferred from the shape/charge complementarity between the T.th. RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of T.th. RuvC homodimer refined at 1.28 Å resolution shows distinct asymmetry near the dimer interface, in the region harboring catalytically important aromatic residues. The observation suggests that the T.th. RuvC homodimer interconverts between two asymmetric conformations, with alternating subunits switched on for DNA strand cleavage. This model provides a structural basis for the 'nick-counter-nick' mechanism in HJ resolution, a mode of HJ processing shared by prokaryotic and eukaryotic HJ resolvases.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 648-656 |
| Number of pages | 9 |
| Journal | Nucleic acids research |
| Volume | 41 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 2013 |
Bibliographical note
Funding Information:We thank the staff of Sector-24 (NE-CAT) of the Advanced Photon Source (APS) for help in x-ray data collection, and Kayo Kurahashi for technical assistance. Computer resources were provided by the Basic Sciences Computing Laboratory of the University of Minnesota Supercomputing Institute. This work is based on research conducted at the APS on the NE-CAT beamlines, which are supported by award RR15301 from the National Center for Research Resources at the National Institutes of Health. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357.
Funding Information:
National Institutes of Health [GM095558 and AI087098 to H.A.]; Undergraduate Research Opportunity Program of the University of Minnesota (to L.C.). Funding for open access charge: NIH.