Generating Crystallographic Models of DNA Dodecamers from Structures of RNase H:DNA Complexes
The DNA dodecamer 5′-d(CGCGAATTCGCG)-3′ is arguably the best studied oligonucleotide and crystal structures of duplexes with this sequence account for a considerable portion of the total number of oligo-2′-deoxynucleotide structures determined over the last 30 years. The dodecamer has commonly served as a template to analyze the effects of sequence on DNA conformation, the conformational properties of chemically modified nucleotides, DNA–ligand interactions as well as water structure and DNA–cation binding. Although molecular replacement is the phasing method of choice given the large number of available models of the dodecamer, this strategy often fails as a result of conformational changes caused by chemical modification, mismatch pairs, or differing packing modes. Here, we describe an alternative approach to determine crystal structures of the dodecamer in cases where molecular replacement does not produce a solution or when crystals of the DNA alone cannot be grown. It is based on the discovery that many dodecamers of the above sequence can be readily co-crystallized with Bacillus halodurans RNase H, whereby the enzyme is unable to cleave the DNA. Determination of the structure of the complex using the protein portion as the search model yields a structural model of the DNA. Provided crystals of the DNA alone are also available, the DNA model from the complex then enables phasing their structures by molecular replacement.
Key wordsDNA Molecular replacement Phasing Protein–DNA interactions Ribonuclease H RNase H
This work was supported by the US National Institutes of Health grant R01 GM055237.
- 10.Egli M, Tereshko V (2004) Lattice- and sequence-dependent binding of Mg2+ in the crystal structure of a B-DNA dodecamer. In: Stellwagen N, Mohanty U (eds) Curvature and deformation of nucleic acids: recent advances, new paradigms, ACS symposium, vol 884. Oxford University Press, New York, pp 87–109Google Scholar
- 13.Nanjunda R, Wilson WD (2012) Binding to the DNA minor groove by heterocyclic dications: from AT-specific monomers to GC recognition with dimers. Curr Protoc Nucleic Acid Chem 51:8.8.1–8.8.20Google Scholar
- 15.Egli M (1998) Towards the structure-based design of nucleic acid therapeutics. In: Weber G (ed) Advances in enzyme regulation, vol 38. Elsevier, Oxford, pp 181–203Google Scholar
- 38.Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive python-based system for macromolecular structure solution. Acta Crystallogr D 66:213–221PubMedCentralPubMedCrossRefGoogle Scholar
- 42.Jiang X, Egli M (2011) Use of chromophoric ligands to visually screen co-crystals of putative protein-nucleic acid complexes. Curr Protoc Nucleic Acid Chem 46:7.15.1–7.15.8Google Scholar