Recognition of 5'-YpG-3' sequences by coupled stacking/hydrogen bonding interactions with amino acid residues

The combined biochemical and structural study of hundreds of protein-DNA complexes has indicated that sequence-specific interactions are mediated by two mechanisms termed direct and indirect readout. Direct readout involves direct interactions between the protein and base-specific atoms exposed in the major and minor grooves of DNA. For indirect readout, the protein recognizes DNA by sensing conformational variations in the structure dependent on nucleotide sequence, typically through interactions with the phosphodiester backbone. Based on our recent structure of Ndt80 bound to DNA in conjunction with a search of the existing PDB database, we propose a new method of sequence-specific recognition that utilizes both direct and indirect readout. In this mode, a single amino acid side-chain recognizes two consecutive base-pairs. The 3'-base is recognized by canonical direct readout, while the 5'-base is recognized through a variation of indirect readout, whereby the conformational flexibility of the particular dinucleotide step, namely a 5'-pyrimidine-purine-3' step, facilitates its recognition by the amino acid via cation-pi interactions. In most cases, this mode of DNA recognition helps explain the sequence specificity of the protein for its target DNA.     

Hydration and recognition of methylated CpG steps in DNA.

The analysis of the hydration pattern around methylated CpG steps in three high resolution (1.7, 2.15 and 2.2 A) crystal structures of A-DNA decamers reveals that the methyl groups of cytosine residues are well hydrated. In comparing the native structure with two structurally distinct forms of the decamer d(CCGCCGGCGG) fully methylated at its CpG steps, this study shows also that in certain structural and sequence contexts, the methylated cytosine base can be more hydrated that the unmodified one. These water molecules seem to be stabilized in front of the methyl group through the formation C-H...O interactions. In addition, these structures provide the first observation of magnesium cations bound to the major groove of A-DNA and reveal two distinct modes of metal binding in methylated and native duplexes. These findings suggest that methylated cytosine bases could be recognized by protein or DNA polar residues through their tightly bound water molecules.     

The nonconserved hinge region and distinct amino-terminal domains of the ROR alpha orphan nuclear receptor isoforms are required for proper DNA bending and ROR alpha-DNA interactions.

ROR alpha 1 and ROR alpha 2 are two isoforms of a novel member of the steroid-thyroid-retinoid receptor superfamily and are considered orphan receptors since their cognate ligand has yet to be identified. These putative receptors have previously been shown to bind as monomers to a DNA recognition sequence composed of two distinct moieties, a 3' nuclear receptor core half-site AGGTCA preceded by a 5' AT-rich sequence. Recognition of this bipartite hormone response element (RORE) requires both the zinc-binding motifs and a group of amino acid residues located at the carboxy-terminal end of the DNA-binding domain (DBD) which is referred to here as the carboxy-terminal extension. In this report, we show that binding of ROR alpha 1 and ROR alpha 2 to the RORE induces a large DNA bend of approximately 130 degrees which may be important for receptor function. The overall direction of the DNA bend is towards the major groove at the center of the 3' AGGTCA half-site. The presence of the nonconserved hinge region which is located between the DBD and the putative ligand-binding domain (LBD) or ROR alpha is required for maximal DNA bending. Deletion of a large portion of the amino-terminal domain (NTD) of the ROR alpha protein does not alter the DNA bend angle but shifts the DNA bend center 5' relative to the bend induced by intact ROR alpha. Methylation interference studies using the NTD-deleted ROR alpha 1 mutant indicate that some DNA contacts in the 5' AT-rich half of the RORE are also shifted 5', while those in the 3' AGGTCA half-site are unaffected. These results are consistent with a model in which the ROR alpha NTD and the nonconserved hinge region orient the zinc-binding motifs and the carboxy-terminal extension of the ROR alpha DBD relative to each other to achieve proper interactions with the two halves of its recognition site. Transactivation studies suggest that both protein-induced DNA bending and protein-protein interactions are important for receptor function.