A simple motif for protein recognition in DNA secondary structures

DNA in a single-stranded form (ssDNA) exists transiently within the cell and comprises the telomeres of linear chromosomes and the genomes of some DNA viruses. As with RNA, in the single-stranded state, some DNA sequences are able to fold into complex secondary and tertiary structures that may be recognized by proteins and participate in gene regulation. To better understand how such DNA elements might fold and interact with proteins, and to compare recognition features to those of a structured RNA, we used in vitro selection to identify ssDNAs that bind an RNA-binding peptide from the HIV Rev protein with high affinity and specificity. The large majority of selected binders contain a non-Watson-Crick G.T base-pair and an adjacent C:G base-pair and both are essential for binding. This GT motif can be presented in different DNA contexts, including a nearly perfect duplex and a branched three-helix structure, and appears to be recognized in large part by arginine residues separated by one turn of an alpha-helix. Interestingly, a very similar GT motif is necessary also for protein binding and function of a well-characterized model ssDNA regulatory element from the proenkephalin promoter.     

A structure-based method for identifying DNA-binding proteins and their sites of DNA-interaction

A classification model of a DNA-binding protein chain was created based on identification of alpha helices within the chain likely to bind to DNA. Using the model, all chains in the Protein Data Bank were classified. For many of the chains classified with high confidence, previous documentation for DNA-binding was found, yet no sequence homology to the structures used to train the model was detected. The result indicates that the chain model can be used to supplement sequence based methods for annotating the function of DNA-binding. Four new candidates for DNA-binding were found, including two structures solved through structural genomics efforts. For each of the candidate structures, possible sites of DNA-binding are indicated by listing the residue ranges of alpha helices likely to interact with DNA.     

Folded chromosome structure and DNA-binding protein of Streptomyces hygroscopicus

The isolation of the folded chromosomal structure from a Streptomyces strain is described. It is shown that the compact DNA structure of Streptomyces hygroscopicus is stabilized by DNA-RNA interactions. Nucleolytic cleavage decreases the sedimentation rate of the originally isolated (membrane-free) folded Streptomyces chromosome from 1500 S to 800 S or 300 S and finally to completely unfolded DNA. Data on ethidium bromide intercalation suggest a dye-induced relaxation and reintroduction of DNA supertwisting. Like naturally occurring covalently closed circular DNA and like the Escherichia coli chromosome, the folded chromosomal DNA of S. hygroscopicus has about one negative superhelix turn per 200 bp. The results further demonstrate that the isolated Streptomyces nucleoid contains a characteristic S protein which exhibits gel electrophoretic properties of a histone-like component similar to that found in E. coli or Thermoplasma acidophilum. Digestion of the nucleoid with proteinase K at 37°C causes elimination of the S protein and unfolding of the compact structure. The S protein is also present in other species of the genus Streptomyces.     

A putative DNA-binding domain in the NUCKS protein

We have studied the DNA-binding properties of a NUCKS-derived, synthetic peptide containing an extended GRP motif. This peptide binds to random-sequence DNA, but did not bind preferentially to poly(dA-dT). A synthetic peptide with the same amino acid composition but with a random sequence did not bind to DNA, suggesting that the structure of the DNA-binding domain plays a pivotal role in the interaction with DNA. NMR and graphic modeling were employed to investigate the structure of the synthetic peptide. It was shown that the DNA-binding peptide constituted an alpha helix in phosphate buffer at pH 5.5. Docking results indicated an almost perfect fit for this small, helical peptide into the major groove of DNA with the possibility of four basic residues interacting with the phosphate backbone of DNA. One consensus site for phosphorylation by Cdk1 is located in the N-terminal end of the DNA-binding peptide. Upon phosphorylation of this site, the binding to DNA was completely prohibited. Immunofluorescence experiments showed that NUCKS was located in the nuclei in proliferating cells in interphase of the cell cycle, but was distributed throughout the cytoplasm in mitotic cells.     

Crp1p,a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae

A synthetic cruciform DNA (X-DNA) was used for screening cellular extracts of Saccharomyces cerevisiae for X-DNA-binding activity. Three X-DNA-binding proteins with apparent molecular mass of 28kDa, 26kDa and 24kDa, estimated by SDS-PAGE, were partially purified. They were identified as N-terminal fragments originating from the same putative protein, encoded by the open reading frame YHR146W, which we named CRP1 (cruciform DNA-recognising protein 1). Expression of CRP1 in Escherichia coli showed that Crp1p is subject to efficient proteolysis at one specific site. Cleavage leads to an N-terminal subpeptide of approximately 160 amino acid residues that is capable of binding specifically X-DNA with an estimated dissociation constant (K(d)) of 800nM, and a C-terminal subpeptide of approximately 305 residues without intrinsic X-DNA-binding activity. The N-terminal subpeptide is of a size similarly to that of the fragments identified in yeast, suggesting that the same cleavage process occurs in the yeast and the E.coli background. This makes the action of a site-specific protease unlikely and favours the possibility of an autoproteolytic activity of Crp1p. The DNA-binding domain of Crp1p was mapped to positions 120-141. This domain can act autonomously as an X-DNA-binding peptide and provides a new, lysine-rich DNA-binding domain different from those of known cruciform DNA-binding proteins (CBPs). As reported earlier for several other CBPs, Crp1p exerts an enhancing effect on the cleavage of X-DNA by endonuclease VII from bacteriophage T4.     

Statistical potential for assessment and prediction of protein structures

Protein structures in the Protein Data Bank provide a wealth of data about the interactions that determine the native states of proteins. Using the probability theory, we derive an atomic distance-dependent statistical potential from a sample of native structures that does not depend on any adjustable parameters (Discrete Optimized Protein Energy, or DOPE). DOPE is based on an improved reference state that corresponds to noninteracting atoms in a homogeneous sphere with the radius dependent on a sample native structure; it thus accounts for the finite and spherical shape of the native structures. The DOPE potential was extracted from a nonredundant set of 1472 crystallographic structures. We tested DOPE and five other scoring functions by the detection of the native state among six multiple target decoy sets, the correlation between the score and model error, and the identification of the most accurate non-native structure in the decoy set. For all decoy sets, DOPE is the best performing function in terms of all criteria, except for a tie in one criterion for one decoy set. To facilitate its use in various applications, such as model assessment, loop modeling, and fitting into cryo-electron microscopy mass density maps combined with comparative protein structure modeling, DOPE was incorporated into the modeling package MODELLER-8.     

Molecular dynamics studies of a DNA-binding protein: 1. A comparison of the trp repressor and trp aporepressor aqueous simulations.

The results of two 30-ps molecular dynamics simulations of the trp repressor and trp aporepressor proteins are presented in this paper. The simulations were obtained using the AMBER molecular mechanical force field and in both simulations a 6-A shell of TIP3P waters surrounded the proteins. The trp repressor protein is a DNA-binding regulatory protein and it utilizes a helix-turn-helix (D helix-turn-E helix) motif to interact with DNA. The trp aporepressor, lacking two molecules of the L-tryptophan corepressor, cannot bind specifically to DNA. Our simulations show that the N- and C-termini and the residues in and near the helix-turn-helix motifs are the most mobile regions of the proteins, in agreement with the X-ray crystallographic studies. Our simulations also find increased mobility of the residues in the turn-D helix-turn regions of the proteins. We find the average distance separating the DNA-binding motifs to be larger in the repressor as compared to the aporepressor. In addition to examining the protein residue fluctuations and deviations with respect to X-ray structures, we have also focused on backbone dihedral angles and corepressor hydrogen-bonding patterns in this paper.