Restriction endonucleases that cleave RNA/DNA heteroduplexes bind dsDNA in A-like conformation
Open Access
- 27 May 2020
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 48 (12), 6954-6969
- https://doi.org/10.1093/nar/gkaa403
Abstract
Restriction endonucleases naturally target DNA duplexes. Systematic screening has identified a small minority of these enzymes that can also cleave RNA/DNA heteroduplexes and that may therefore be useful as tools for RNA biochemistry. We have chosen AvaII (G↓GWCC, where W stands for A or T) as a representative of this group of restriction endonucleases for detailed characterization. Here, we report crystal structures of AvaII alone, in specific complex with partially cleaved dsDNA, and in scanning complex with an RNA/DNA hybrid. The specific complex reveals a novel form of semi-specific dsDNA readout by a hexa-coordinated metal cation, most likely Ca2+ or Mg2+. Substitutions of residues anchoring this non-catalytic metal ion severely impair DNA binding and cleavage. The dsDNA in the AvaII complex is in the A-like form. This creates space for 2′-OH groups to be accommodated without intra-nucleic acid steric conflicts. PD-(D/E)XK restriction endonucleases of known structure that bind their dsDNA targets in the A-like form cluster into structurally similar groups. Most such enzymes, including some not previously studied in this respect, cleave RNA/DNA heteroduplexes. We conclude that A-form dsDNA binding is a good predictor for RNA/DNA cleavage activity.Funding Information
- Ministry of Science and Higher Education (0295/B/PO1/2008/34, N N301 425038, N N301 028 934)
- National Science Centre (UMO-2011/02/A/NZ1/00052, UMO-2014/13/B/NZ1/03991, UMO-2014/14/M/NZ5/00558)
- European Regional Development Fund (TEAM TECH CORE FACILITY/2017–3/4, POIR.04.04.00–00-31DF/17–00)
- IIMCB
This publication has 70 references indexed in Scilit:
- Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteriaProceedings of the National Academy of Sciences of the United States of America, 2012
- RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein ComplexCell, 2009
- REBASE—a database for DNA restriction and modification: enzymes, genes and genomesNucleic Acids Research, 2009
- Crystal structure of the ββα-Me type II restriction endonuclease Hpy99I with target DNANucleic Acids Research, 2009
- Intrinsic Dynamics of Restriction Endonuclease EcoO109I Studied by Molecular Dynamics Simulations and X-Ray Scattering Data AnalysisBiophysical Journal, 2009
- Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7Nature Protocols, 2008
- Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analysesNucleic Acids Research, 2008
- Version 1.2 of the Crystallography and NMR systemNature Protocols, 2007
- Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetricallyNucleic Acids Research, 2007
- Gapped BLAST and PSI-BLAST: a new generation of protein database search programsNucleic Acids Research, 1997