Thermodynamic basis for sequence-specific recognition of ssDNA by an autoantibody

11F8 is a sequence-specific DNA binding monoclonal autoantibody previously isolated from an autoimmune lupus-prone mouse [Stevens, S. Y., and Glick, G. D. (1999) Biochemistry 38, 560-568]. This antibody, like many other lupus anti-DNAs, localizes to kidney tissue and eventually leads to renal damage through a process that first involves the binding of DNA antigens. A series of experiments were conducted to investigate the thermodynamic and structural basis by which this antibody discriminates between specific, noncognate, and nonspecific sequences. Sequence-specific binding occurs with a minimal dependence on the polyelectrolyte effect along with a favorable binding enthalpy reflecting the presence of base stacking and contacts to DNA bases. This favorable binding enthalpy apparently is derived from desolvation at the binding interface and is consistent with recent models of the nonclassical hydrophobic effect. Noncognate recognition is also driven by the nonclassical hydrophobic effect, but is accompanied by highly unfavorable entropies that are responsible for reduced affinity relative to the high-affinity consensus sequence. Nonspecific recognition is driven completely by the polyelectrolyte effect involving extensive electrostatic interactions with the phosphate backbone. Collectively, the data demonstrate the ability of 11F8 to adapt its mode of binding to the available DNA surface and provide a thermodynamic model for sequence-specific recognition of single-stranded DNA. The salient features of this model employ the paradigms invoked to explain protein.dsDNA, protein.RNA, and antibody.antigen binding.     

Role of conformational dynamics in sequence-specific autoantibody*ssDNA recognition

11F8 is a sequence-specific monoclonal anti-ssDNA autoantibody isolated from a lupus prone mouse that forms pathogenic complexes with ssDNA, resulting in kidney damage. Prior studies show that specificity is mediated by a somatic mutation from serine at (31)V(H) to arginine. Reversion back to serine in 11F8 resulted in >30-fold decrease in affinity and altered thermodynamic and kinetic parameters for sequence-specific recognition of its cognate ssDNA ligand. Mutagenesis and structural studies suggest that (R31)V(H) contacts ssDNA via a salt bridge and a bidentate hydrogen bond and may further contribute to specificity by altering binding-site conformation. Fluorescence resonance energy transfer experiments were conducted to assess the kinetics of conformational change during 11F8*ssDNA association. The extent of rearrangement between the six complementary determining regions in the 11F8*ssDNA complex with germline serine or somatically mutated arginine at residue 31 of the heavy chain was examined. Our studies show that greater conformational change occurs in five of six complementarity determining regions after the heavy chain germline J558 sequence undergoes mutation to arginine at (31)V(H).     

Mutational analysis of a sequence-specific ssDNA binding lupus autoantibody

11F8 is a murine anti-ssDNA monoclonal autoantibody isolated from a lupus prone autoimmune mouse. This mAb binds sequence specifically, and prior studies have defined the thermodynamic and kinetic basis for sequence-specific recognition of ssDNA (Ackroyd, P. C., et al. (2001) Biochemistry 40, 2911-2922; Beckingham, J. A. and Glick, G. D. (2001) Bioorg. Med. Chem. 9, 2243-2252). Here we present experiments designed to identify the residues on 11F8 that mediate sequence-specific, noncognate, and nonspecific recognition of ssDNA and their contribution to the overall binding thermodynamics. Site-directed mutagenesis of an 11F8 single-chain construct reveals that six residues within the complementarity determining regions of 11F8 account for ca. 80% of the binding free energy and that there is little cooperativity between these residues. Germline-encoded aromatic and hydrophobic side chains provides the basis for nonspecific recognition of single-stranded thymine nucleobases. Sequence-specific recognition is controlled by a tyrosine in the heavy chain along with a somatically mutated arginine residue. Our data show that the manner in which 11F8 achieves sequence-specific recognition more closely resembles RNA-binding proteins such as U1A than other types of nucleic acid binding proteins. In addition, comparing the primary sequence of 11F8 with clonally related antibodies that differ by less than five amino acids suggests that somatic mutations which confer sequence specificity may be a feature that distinguishes glomerulotrophic pathogenic anti-DNA from those that are benign.     

Sequence specific recognition of ssDNA by a lupus autoantibody: kinetics and mechanism of binding.

11F8 is a pathogenic anti-ssDNA monoclonal autoantibody isolated from a lupus-prone mouse. Previous studies have established that 11F8 is sequence specific. To determine the basis for the observed binding specificity, stopped-flow fluorescence spectroscopy was used to measure the kinetic parameters and establish the mechanisms for the association of 11F8 with its target sequence, noncognate, and nonspecific ssDNA ligands. The data revealed that sequence-specific binding follows a two-step mechanism where the initial association step is second order. Values of k(1) are fast and above the modified Smoluchowski limit for a diffusion limited interaction (10(5)-10(6)M(-1)s(-1)). The dependency of k(1) on [salt] and solvent polarity indicates that electrostatic steering is responsible for this rapid association rate. The second association step is rate limiting and is characteristic of an isomerization process during which binding interfaces are optimized. This step apparently is driven by the desolvation of hydrophobic surfaces within the binding interface. The differences in the rate of dissociation for the various DNA ligands suggest that specificity is governed primarily through the dissociation of the final complexes.     

Porphyrin-linked oligonucleotides. Synthesis and sequence-specific modification of ssDNA

Oligonucleotide derivatives bearing hemin and deuterohemin groups were synthesized. The derivatives efficiently react with the complementary nucleotide sequence in ssDNA forming covalent adducts and piperidine-labile sites. In the case of the deuterohemin derivative, some direct cleavage of the target DNA occurs.     

Molecular biological analysis of sequence-specific DNA recognition and cleavage by metallobleomycins

The DNase I footprinting analysis shows binding sites of approximately two or three base pairs, in particular 5'-XGC sequences, for the green-colored Co(III) and fully oxidized Fe(III) complexes of bleomycin (BLM). In contrast to covalent attachment of guanine N-7 with aflatoxin B1 or dimethyl sulfate, the modification of guanine 2-amino group with anthramycin remarkably inhibits the DNA cleavages at 5'-GC and 5'-GT sites by the iron and cobalt complex systems of BLM. The present results strongly indicate that metallobleomycin binds in minor groove of B-DNA and that the 2-amino group of guanine adjacent to 5'-side of the cleaved pyrimidine base is one key element of specific 5'-GC or 5'-GT recognition by metallobleomycin. On the basis of these experimental data, possible binding mode of metallobleomycin in B-DNA helix has been proposed by computer-constructed model building.     

Themes in ssDNA recognition by telomere-end protection proteins

The ends of eukaryotic linear chromosomes are unique structures that require special management by the cell. If left unattended, the ends are inappropriately processed, leading to genomic instability and problems with proliferation. Telomeres are specialized nucleoprotein structures that restore chromosome stability by protecting and maintaining chromosome ends. Proper telomere function is facilitated, in part, by the telomere-end protection (TEP) family of proteins, which targets the 3' single-stranded (ss) overhang region of the telomere via a specialized ssDNA-binding domain (DBD). With the recent availability of the structures of these DBDs, the ssDNA-binding characteristics of TEP proteins can be compared and the common underlying mechanisms of ssDNA recognition identified, thus providing insights into telomere function.     

RNA recognition and cleavage by sequence-specific endoribonucleases

Inactivation of RNA molecules by sequence-specific endoribonucleolytic cleavage is a subtle mechanism by which cells regulate gene expression. Sequence-specific endoribonucleases can recognize and cleave particular phosphodiester bonds confined within hundreds/thousands of chemically similar bonds. Here, we present a comparative analysis of the mechanisms used by endoribonucleases to select and cleave their target RNA molecules. This analysis is based on the very recent molecular details obtained from the structural and/or biochemical studies of nine sequence-specific ribonucleases that target messenger, ribosomal, and transfer RNA molecules. This analysis shows that despite the absence of sequence homologies and the wide diversity of biological sources (prokaryotes, archaea and eukaryotes), the sequence-specific ribonucleases studied here adopt limited structural folds, catalyze their cleavage reactions using a common chemistry and involve a very limited set of amino acids for both RNA binding and processing.     

Multiple-step kinetic mechanisms of the ssDNA recognition process by human polymerase beta in its different ssDNA binding modes

The kinetics of human polymerase beta (pol beta) binding to the single-stranded DNA, in the (pol beta)(16) and (pol beta)(5) binding modes, that differ in the number of occluded nucleotide residues in the protein-DNA complexes, have been examined, using the fluorescence stopped-flow technique. This is the first determination of the mechanism of ssDNA recognition by human pol beta. Binding of the enzyme to the ssDNA containing fluorescein in the place of one of the nucleotides is characterized by a strong DNA fluorescence increase, providing the required signal to quantitatively examine the complex mechanism of ssDNA recognition. The experiments were performed with the ssDNA 20-mer, which engages the polymerase in the (pol beta)(16) binding mode and encompasses the total DNA-binding site of the enzyme, and with the 10-mer, which exclusively forms the (pol beta)(5) binding mode engaging only the 8-kDa domain of the enzyme. The obtained data and analyses indicate that the (pol beta)(16) formation occurs by a minimum four-step, sequential mechanism: (reaction: see text). Formation of the (pol beta)(5) binding mode proceeds with the same mechanism; however, both binding modes differ in the energetics of the partial reactions and the structure of the intermediates. Quantitative amplitude analysis, using the matrix projection operator approach, allowed us to determine molar fluorescence intensities of all intermediates relative to the fluorescence of the free DNA. The results indicate that (pol beta)(16) binding mode formation, which is initiated by the association of the 8-kDa domain with the DNA, is followed by subsequent intermediates stabilized by DNA binding to the 31-kDa domain. Comparison with the (pol beta)(5) binding mode formation indicates that transitions of the enzyme-DNA complex in both modes are induced at the interface of the 8-kDa domain and the DNA. The sequential nature of the mechanism indicates the lack of a conformational preequilibrium of the enzyme prior to ssDNA binding.     

DNA determinants in sequence-specific recognition by XmaI endonuclease.

The XmaI endonuclease recognizes and cleaves the sequence C decreases CCGGG. Magnesium is required for catalysis, however, the enzyme forms stable, specific complexes with DNA in the absence of magnesium. An association constant of 1.2 x 10(9)/M was estimated for the affinity of the enzyme for a specific 195 bp fragment. Competition assays revealed that the site-specific association constant represented an approximately 10(4)-fold increase in affinity over that for non-cognate sites. Missing nucleoside analyses suggested an interaction of the enzyme with each of the cytosines and guanines within the recognition site. Recognition of each of the guanines was also indicated by dimethylsulfate interference footprinting assays. The phosphates 5' to the guanines within the recognition site appeared to be the major sites of interaction of XmaI with the sugar-phosphate backbone. No significant interaction of the protein was observed with phosphates flanking the recognition sequence. Comparison of the footprinting patterns of XmaI with those of the neoschizomer SmaI (CCC decreases GGG) revealed that the two enzymes utilize the same DNA determinants in their specific interaction with the CCCGGG recognition site.     

Origin of sequence-specific recognition of DNA by non-intercalating anti-tumor antibiotics

Partitioning of energy in the interaction of non-intercalating antibiotics (netropsin, netropsin without its cationic ends and two analogs of distamycin A) with different base sequences of B-DNA is studied here by the atom-atom potential technique and geometry optimization procedures. The results show that electrostatic forces contribute substantially to the stabilization energy as well as to the sequence specificity. The hydrogen-bonding term is also sequence specific and is significant in properly orienting the drug molecule. Relative roles of the hydrogen bonding and electrostatic interactions depend on the dielectric property of the medium.     

Molecular basis of sequence-specific recognition of pre-ribosomal RNA by nucleolin

The structure of the 28 kDa complex of the first two RNA binding domains (RBDs) of nucleolin (RBD12) with an RNA stem-loop that includes the nucleolin recognition element UCCCGA in the loop was determined by NMR spectroscopy. The structure of nucleolin RBD12 with the nucleolin recognition element (NRE) reveals that the two RBDs bind on opposite sides of the RNA loop, forming a molecular clamp that brings the 5' and 3' ends of the recognition sequence close together and stabilizing the stem-loop. The specific interactions observed in the structure explain the sequence specificity for the NRE sequence. Binding studies of mutant proteins and analysis of conserved residues support the proposed interactions. The mode of interaction of the protein with the RNA and the location of the putative NRE sites suggest that nucleolin may function as an RNA chaperone to prevent improper folding of the nascent pre-rRNA.     

Probing the mechanism of recognition of ssDNA by the Cdc13-DBD

The Saccharomyces cerevisiae protein Cdc13 tightly and specifically binds the conserved G-rich single-stranded overhang at telomeres and plays an essential role in telomere end-protection and length regulation. The 200 residue DNA-binding domain of Cdc13 (Cdc13-DBD) binds an 11mer single-stranded representative of the yeast telomeric sequence [Tel11, d(GTGTGGGTGTG)] with a 3 pM affinity and specificity for three bases (underlined) at the 5' end. The structure of the Cdc13-DBD bound to Tel11 revealed a large, predominantly aromatic protein interface with several unusual features. The DNA adopts an irregular, extended structure, and the binding interface includes a long ( approximately 30 amino acids) structured loop between strands beta2-beta3 (L(2-3)) of an OB-fold. To investigate the mechanism of ssDNA binding, we studied the free and bound states of Cdc13-DBD using NMR spectroscopy. Chemical shift changes indicate that the basic topology of the domain, including L(2-3), is essentially intact in the free state. Changes in slow and intermediate time scale dynamics, however, occur in L(2-3), while conformational changes distant from the DNA interface suggest an induced fit mechanism for binding in the 'hot spot' for binding affinity and specificity. These data point to an overall binding mechanism well adapted to the heterogeneous nature of yeast telomeres.     

Nucleotide preferences in sequence-specific recognition of DNA by c-myb protein.

Using a binding site selection procedure, we have found that sequence-specific DNA-binding by the mouse c-myb protein involves recognition of nucleotides outside of the previously identified hexanucleotide motif. Oligonucleotides containing a random nucleotide core were immunoprecipitated in association with c-Myb, amplified by the Polymerase Chain Reaction and cloned in plasmids prior to sequencing. By alignment of sequences it was apparent that additional preferences existed at each of three bases immediately 5' of the hexanucleotide consensus, allowing an extension of the preferred binding site to YGRCVGTTR. The contributions of these 5' nucleotides to binding affinity was established in bandshift analyses with oligonucleotides containing single base substitutions; in particular, it was found that replacement of the preferred guanine at position -2 with any other base greatly reduced c-Myb binding. We found that the protein encoded by the related B-myb gene bound the preferred c-Myb site with similar affinity; however, B-Myb and c-Myb showed distinct preferences for the identity of the nucleotide at position -1 relative to the hexanucleotide consensus. This study demonstrates that the c-Myb DNA-binding site is more extensive than recognised hitherto and points to similar but distinct nucleotide preferences in recognition of DNA by related Myb proteins.     

Electron microscopic studies of sequence-specific recognition of duplex DNA by different ligands

The following ligands were used to study sequence specific recognition of duplex DNA by electron microscopic techniques: methyltransferases BspR1 and EcoR124 (recognition sequences GGCC and GAAN7RTCG, respectively), a biotinylated deoxyoligonucleotide 5′-CTCTCTCTCTCTCT-3′ capable of forming triplex DNA, and PNA oligomer H-T₁₀-LysNH₂. For each ligand the best conditions for electron microscopic (EM)detection of stable specific complex formation were determined. It was demonstrated that EM allowed us to determine the position of the individual target site with an error of 15–20 bp, the relative affinities for individual target sites and kinetic parameters of the binding. These results open new possibilities for EM investigations of sequence-specific interactions with a wide range of other ligands of a similar nature. They also imply that a wide range of different sequences can be unambiguously and precisely mapped by EM and greatly extend the scope of EM applications for physical mapping of genomic DNA.     

Kinetic analysis of enzyme inactivation by an autodecaying reagent

A simple method is described for the determination of both the pseudo-first-order rate constant and the second-order rate constant for enzyme inactivation by a chemical reagent which itself undergoes exponential decay. The validity of this method has been demonstrated in two test cases in which the labile diethyl pyrocarbonate was used to inactivate salicylate hydroxylase and bacterial luciferase.