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V B Zhurkin

Publications and source records attributed to V B Zhurkin.

At least 19 recordsLinked to original sources
PubMed2001

Structural basis for SRY-dependent 46-X,Y sex reversal: modulation of DNA bending by a naturally occurring point mutation.

The HMG-box domain of the human male sex-determining factor SRY, hSRY(HMG) (comprising residues 57-140 of the full-length sequence), binds DNA sequence-specifically in the minor groove, resulting in substantial DNA bending. The majority of point mutations resulting in 46X,Y sex reversal are located within this domain. One clinical de novo mutation, M64I in the full-length hSRY sequence, which corresponds to M9I in the present hSRY(HMG) construct, acts principally by reducing the extent of DNA bending. To elucidate the structural consequences of the M9I mutation, we have solved the 3D solution structures of wild-type and M9I hSRY(HMG) complexed to a DNA 14mer by NMR, including the use of residual dipolar couplings to derive long-range orientational information. We show that the average bend angle (derived from an ensemble of 400 simulated annealing structures for each complex) is reduced by approximately 13 degrees from 54(+/-2) degrees in the wild-type complex to 41(+/-2) degrees in the M9I complex. The difference in DNA bending can be localized directly to changes in roll and tilt angles in the ApA base-pair step involved in interactions with residue 9 and partial intercalation of Ile13. The larger bend angle in the wild-type complex arises as a direct consequence of steric repulsion of the sugar of the second adenine by the bulky S(delta) atom of Met9, whose position is fixed by a hydrogen bond with the guanidino group of Arg17. In the M9I mutant, this hydrogen bond can no longer occur, and the less bulky C(gamma)m methyl group of Ile9 braces the sugar moieties of the two adenine residues, thereby decreasing the roll and tilt angles at the ApA step by approximately 8 degrees and approximately 5 degrees, respectively, and resulting in an overall difference in bend angle of approximately 13 degrees between the two complexes. To our knowledge, this is one of the first examples where the effects of a clinical mutation involving a protein-DNA complex have been visualized at the atomic level.

Amino Acid Motifs↗
PubMed2001

Sequence-dependent B<-->A transition in DNA evaluated with dimeric and trimeric scales.

Experimental data on the sequence-dependent B<-->A conformational transition in 24 oligo- and polymeric duplexes yield optimal dimeric and trimeric scales for this transition. The 10 sequence dimers and the 32 trimers of the DNA duplex were characterized by the free energy differences between the B and A forms in water solution. In general, the trimeric scale describes the sequence-dependent DNA conformational propensities more accurately than the dimeric scale, which is likely related to the trimeric model accounting for the two interfaces between adjacent base pairs on both sides (rather than only one interface in the dimeric model). The exceptional preference of the B form for the AA:TT dimers and AAN:N'TT trimers is consistent with the cooperative interactions in both grooves. In the minor groove, this is the hydration spine that stabilizes adenine runs in B form. In the major groove, these are hydrophobic interactions between the thymine methyls and the sugar methylene groups from the preceding nucleotides, occurring in B form. This interpretation is in accord with the key role played by hydration in the B<-->A transition in DNA. Importantly, our trimeric scale is consistent with the relative occurrences of the DNA trimers in A form in protein-DNA cocrystals. Thus, we suggest that the B/A scales developed here can be used for analyzing genome sequences in search for A-philic motifs, putatively operative in the protein-DNA recognition.

Carbohydrates↗
PubMed2001

Gal repressosome contains an antiparallel DNA loop.

Gal repressosome assembly and repression of the gal operon in Escherichia coli occurs when two dimeric GalR proteins and the histone-like HU protein bind to cognate sites causing DNA looping. Structure-based genetic analysis defined the GalR surfaces interacting to form a stacked, V-shaped, tetrameric structure. Stereochemical models of the four possible DNA loops compatible with the GalR tetramer configuration were constructed using the sequence-dependent structural parameters of the interoperator DNA and conformation changes caused by GalR and asymmetric HU binding. Evaluation of their DNA elastic energies gave unambiguous preference to a loop structure in which the two gal operators adopt an antiparallel orientation causing undertwisting of DNA.

Amino Acid Substitution↗
PubMed2000

Radioprobing of a RecA-three-stranded DNA complex with iodine 125: evidence for recognition of homology in the major groove of the target duplex.

A fundamental problem in homologous recombination is how homology between DNAs is recognized. In all current models, a recombination protein loads onto a single strand of DNA and scans another duplex for homology. When homology is found, a synaptic complex is formed, leading to strand exchange and a heteroduplex. A novel technique based on strand cleavage by the Auger radiodecay of iodine 125, allows us to determine the distances between (125)I on the incoming strand and the target sugars of the duplex DNA strands in an Escherichia coli RecA protein-mediated synaptic complex. Analysis of these distances shows that the complex represents a post-strand exchange intermediate in which the heteroduplex is located in the center, while the outgoing strand forms a relatively wide helix intertwined with the heteroduplex and located in its minor groove. The structure implies that homology is recognized in the major groove of the duplex.

Base Sequence↗
PubMed2000

Modeling DNA deformations.

Recent developments have been made in modeling double-helical DNA at four levels of three-dimensional structure: the all-atom level, whereby an oligonucleotide duplex is surrounded by a shroud of solvent molecules; the base-pair level, with explicit backbone atoms; the mesoscopic level, that is, a few hundred base pairs, with the local duplex conformation described by knowledge-based harmonic energy functions; and the scale of several thousand nucleotides, with the duplex described as an ideal elastic rod. Predictions of the sequence-dependent bending and twisting of the double helix, as well as solvent- and force-induced B-->A and over-stretching conformational transitions, are compared with experimental data. These subtle conformational changes are critical to the functioning of the double helix, including its packaging in the close confines of the cell, the mutual fit of DNA and protein in nucleoprotein complexes, and the effective recognition of base pairs in recombination and transcription.

Animals↗
PubMed1999

DNA stretching and compression: large-scale simulations of double helical structures.

Computer-simulated elongation and compression of A - and B -DNA structures beyond the range of thermal fluctuations provide new insights into high energy "activated" forms of DNA implicated in biochemical processes, such as recombination and transcription. All-atom potential energy studies of regular poly(dG).poly(dC) and poly(dA).poly(dT) double helices, stretched from compressed states of 2.0 A per base-pair step to highly extended forms of 7.0 A per residue, uncover four different hyperfamilies of right-handed structures that differ in mutual base-pair orientation and sugar-phosphate backbone conformation. The optimized structures embrace all currently known right-handed forms of double-helical DNA identified in single crystals as well as non-canonical forms, such as the original "Watson-Crick" duplex with trans conformations about the P-O5' and C5'-C4' backbone bonds. The lowest energy minima correspond to canonical A and B -form duplexes. The calculations further reveal a number of unusual helical conformations that are energetically disfavored under equilibrium conditions but become favored when DNA is highly stretched or compressed. The variation of potential energy versus stretching provides a detailed picture of dramatic conformational changes that accompany the transitions between various families of double-helical forms. In particular, the interchanges between extended canonical and non-canonical states are reminiscent of the cooperative transitions identified by direct stretching experiments. The large-scale, concerted changes in base-pair inclination, brought about by changes in backbone and glycosyl torsion angles, could easily give rise to the observed sharp increase in force required to stretch single DNA molecules more than 1.6-1.65 times their canonical extension. Our extended duplexes also help to tie together a number of previously known structural features of the RecA-DNA complex and offer a self-consistent stereochemical model for the single-stranded/duplex DNA recognition brought in register by recombination proteins. The compression of model duplexes, by contrast, yields non-canonical structures resembling the deformed steps in crystal complexes of DNA with the TATA-box binding protein (TBP). The crystalline TBP-bound DNA steps follow the calculated compression-elongation pattern of an unusual "vertical" duplex with base planes highly inclined with respect to the helical axis, exposed into the minor groove, and accordingly accessible for recognition.Significantly, the double helix can be stretched by a factor of two and compressed roughly in half before its computed internal energy rises sharply. The energy profiles show that DNA extension-compression is related not only to the variation of base-pair Rise but also to concerted changes of Twist, Roll, and Slide. We suggest that the high energy "activated" forms calculated here are critical for DNA processing, e.g. nucleo-protein recognition, DNA/RNA synthesis, and strand exchange.

Computer Simulation↗
PubMed1999

p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting.

DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA. By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53. Our data show that four subunits of p53DBD bend the DNA by 32-36 degrees, whereas wt p53 bends it by 51-57 degrees. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by approximately 35 degrees and approximately 70 degrees, respectively. These results are in accord with molecular modeling studies of the tetrameric complex. Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53-DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers. This bending is consistent with the inherent sequence-dependent anisotropy of the duplex. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding. The novel architecture of the p53 tetrameric complex has important functional implications including possible p53 interactions with chromatin.

Base Sequence↗
PubMed1999

Detecting the DNA kinks in a DNA-CRP complex in solution with iodine-125 radioprobing.

Auger-electron-emitting radioisotopes such as 125I produce DNA strand breaks within nanometer range of the decay site. Here we analyze these breaks in order to study changes in DNA conformation upon binding with cyclic AMP receptor protein (CRP) in solution. The clear difference we found in break frequency in the CRP-DNA complex, as compared to the naked DNA duplex, correlates with the increased distances between the deoxyriboses and the radioiodine atom caused by the CRP-induced kink observed in the cocrystal. Thus, we demonstrate that 125I radioprobing can be used to study fine conformational changes of DNA within DNA-protein complexes.

Base Sequence↗
PubMed1998

DNA sequence-dependent deformability deduced from protein-DNA crystal complexes.

The deformability of double helical DNA is critical for its packaging in the cell, recognition by other molecules, and transient opening during biochemically important processes. Here, a complete set of sequence-dependent empirical energy functions suitable for describing such behavior is extracted from the fluctuations and correlations of structural parameters in DNA-protein crystal complexes. These elastic functions provide useful stereochemical measures of the local base step movements operative in sequence-specific recognition and protein-induced deformations. In particular, the pyrimidine-purine dimers stand out as the most variable steps in the DNA-protein complexes, apparently acting as flexible "hinges" fitting the duplex to the protein surface. In addition to the angular parameters widely used to describe DNA deformations (i.e., the bend and twist angles), the translational parameters describing the displacements of base pairs along and across the helical axis are analyzed. The observed correlations of base pair bending and shearing motions are important for nonplanar folding of DNA in nucleosomes and other nucleoprotein complexes. The knowledge-based energies also offer realistic three-dimensional models for the study of long DNA polymers at the global level, incorporating structural features beyond the scope of conventional elastic rod treatments and adding a new dimension to literal analyses of genomic sequences.

DNA↗
PubMed1998

A role for CH...O interactions in protein-DNA recognition.

The concept of CH...O hydrogen bonds has recently gained much interest, with a number of reports indicating the significance of these non-classical hydrogen bonds in stabilizing nucleic acid and protein structures. Here, we analyze the CH...O interactions in the protein-DNA interface, based on 43 crystal structures of protein-DNA complexes. Surprisingly, we find that the number of close intermolecular CH...O contacts involving the thymine methyl group and position C5 of cytosine is comparable to the number of protein-DNA hydrogen bonds involving nitrogen and oxygen atoms as donors and acceptors. A comprehensive analysis of the geometries of these close contacts shows that they are similar to other CH...O interactions found in proteins and small molecules, as well as to classical NH...O hydrogen bonds. Thus, we suggest that C5 of cytosine and C5-Met of thymine form relatively weak CH...O hydrogen bonds with Asp, Asn, Glu, Gln, Ser, and Thr, contributing to the specificity of recognition. Including these interactions, in addition to the classical protein-DNA hydrogen bonds, enables the extraction of simple structural principles for amino acid-base recognition consistent with electrostatic considerations.

Base Composition↗
PubMed1997

Architectural accommodation in the complex of four p53 DNA binding domain peptides with the p21/waf1/cip1 DNA response element.

High resolution chemical footprinting and cross-linking experiments have provided a basis for elucidating the overall architecture of the complex between the core DNA binding domain of p53 (p53DBD, amino acids 98-309) and the p21/waf1/cip1 DNA response element implicated in the G1/S phase cell cycle checkpoint. These studies complement both a crystal structure and earlier biophysical studies and provide the first direct experimental evidence that four subunits of p53DBD bind to the response element in a regular staggered array having pseudodyad symmetry. The invariant guanosines in the highly conserved C(A/T)|(T/A)G parts of the consensus half-sites are critical to the p53DBD-DNA binding. Molecular modeling of the complex using the observed peptide-DNA contacts shows that when four subunits of p53DBD bind the response element, the DNA has to bend approximately 50 degrees to relieve steric clashes among different subunits, consistent with recent DNA cyclization studies. The overall lateral arrangement of the four p53 subunits with respect to the DNA loop comprises a novel nucleoprotein assembly that has not been reported previously in other complexes. We suggest that this kind of nucleoprotein superstructure may be important for p53 binding to response elements packed in chromatin and for subsequent transactivation of p53-mediated genes.

Base Sequence↗
PubMed1995

Probing the structure of a putative intermediate in homologous recombination: the third strand in the parallel DNA triplex is in contact with the major groove of the duplex.

A three-stranded DNA that is a putative intermediate of homologous recombination is a novel DNA triplex, R-form DNA. In R-form DNA the third strand includes both purines and pyrimidines and is parallel to the identical strand of the duplex. To test and refine our previously proposed R-form base triplets we have used two approaches: (1) dimethyl sulfate protection of R-form DNA; and (2) thermal dissociation of R-form DNAs in which the duplex strands were substituted in a strand-specific manner with either 7-deaza-guanine or 7-deaza-adenine. Together, the footprinting and isosteric substitution results demonstrate that the third strand in R-form DNA is in contact with the purines in the N7 position in the major groove of the Watson-Crick duplex in three ((GC):G, (AT):A and (TA):T) out of the four possible triplets. Furthermore, these results suggest that the N7 positions of the duplex play a significant role in stabilizing the DNA-DNA contacts during the homology recognition process.

Bacteriophage M13↗
PubMed1995

B-DNA twisting correlates with base-pair morphology.

The observed sequence dependence of the mean twist angles in 38 B-DNA crystal structures can be understood in terms of simple geometrical features of the constituent base-pairs. Structures with low twist appear to unwind in response to severe steric clashes of large exocyclic groups (such as NH2-NH2) in the major and minor grooves, while those with high twist are subjected to lesser contacts (H-O and H-H). We offer a simple clash function that depends on base-pair morphology (i.e. the chemical constitution of base-pairs) and satisfactorily accounts for the twist angles of the ten common Watson-Crick dimer steps both in the solid state and in solution. The twist-clash correlation that we find here still holds when extended to modified bases. In addition to Calladine's purine-purine clashes, we add other close contacts between bases in the grooves, and consider the conformational restrictions on the geometry of the sugar-phosphate backbone (namely, we emphasize the tendency of DNA to conserve virtual backbone length). The significance of this finding is threefold: (1) sequence-dependent DNA twisting is directly involved in protein-DNA interactions; (2) strong correlation between Twist and Roll helps to elucidate the bending of the double helix as a function of base sequence; (3) it is possible to anticipate the effects of chemical modifications on twisting and bending. The mutual correlations of other structural parameters with the twist make this angle a primary determinant of DNA conformational heterogeneity.

Base Sequence↗
PubMed1995

CRP-DNA complexes: inducing the A-like form in the binding sites with an extended central spacer.

The consensus DNA sequence for binding of the Escherichia coli cyclic AMP receptor protein (CRP) has two symmetrically related inverted recognition elements TGTGA:TCACA, separated by a variable spacer, normally 6 bp long. We have shown that the CRP-cAMP complex, when bound to synthetic binding sites with an extended 8 bp spacer segment, induces an increase in the DNA circular dichroism (CD). The CD change at lambda > 275 nm agrees with the shift of approximately one helical turn of DNA into A-like form. The B-conformation is preserved for CRP binding sites similar to that in the lac and uxaCA promoters with 6 bp spacers. Another effect accompanying DNA binding is a dramatic increase of the negative CD magnitude in the spectral region of the ligand cAMP, at lambda < 272 nm. This effect is observed when CRP binds to specific sites with 6 or 8 bp spacers as well as to non-specific DNA. We reason that the A-like form arises by compressing and unwinding the DNA in CRP-DNA complexes having 8 bp central spacers. This serves to maintain a fixed length and twisting angle and is controlled by the protein's relatively rigid frame. This model is consistent with the observation that some binding sites with 6 bp spacers may also show the CD increase inherent to the sites with the extended 8 bp spacers. These 6 bp spacers are characterized by an increased twisting angle that requires their unwinding to bind to CRP. We propose that a mutual adaptation between CRP and binding sites by local untwisting and a B-->A-like transition in the DNA is of general importance and may occur in other protein-DNA complexes, such as the complex of RNA polymerase with promoter DNA.

Base Sequence↗
PubMed1994

A parallel DNA triplex as a model for the intermediate in homologous recombination.

Parallel DNA triplexes considered to be putative intermediates in homologous recombination, are studied by means of theoretical conformational analysis. These triplexes are denoted as the R-form DNA. Two types of triplexes are analyzed: extended R-form DNA, modeling the triple standard structure, created transiently in the presence of recombination proteins (e.g. RecA); and collapsed R-form, obtained after deproteinization. These structures are stereochemically possible for any arbitrary sequence and have the following properties: (1) the third, R-strand, is parallel to the identical duplex strand and is located in the major groove of the duplex; (2) positions of all four bases in the R-strand are nearly isomorphic; (3) the proposed triplets are consistent with the chemical modification data for deproteinized DNA; we suggest, however, that they are the same in the RecA-DNA complex as well. Since the patterns of charges on each base of the R-strand are strictly complementary to the charges of the homologous Watson-Crick (WC) pair in the major groove, we propose that the selection of the homologous sequence may occur through these complementary electrostatic interactions (electrostatic recognition code). We demonstrate that in the collapsed triplex with a rise of about 3.4 A, the bases from the third R-strand can be inclined and interact with two WC base-pairs simultaneously, which could lead to recognition errors. These mispairings are unlikely in the extended triplex. Therefore, we speculate that a functional role of the extended and underwound DNA structure, transiently formed in the complex with RecA protein, is to obviate such errors and increase the stringency of recognition. In other words, RecA plays the role of a DNA chaperone facilitating the recognition of the single stranded DNA and the duplex. Finally, we show that the proposed isomorphic triplets are conformationally advantageous for strand exchange.

Base Composition↗
PubMed1993

CRP-binding sites: evidence for two structural classes with 6-bp and 8-bp spacers.

While classifying protein binding DNA sequences of the type GTGNxCAC, based on the size of Nx [Shumilov, Mol. Biologya (Engl. Transl.) 21 (1987) 168-187], we had previously found that the cyclic AMP receptor protein (CRP)-binding sites found in the Escherichia coli genome are of at least two classes: (i) those with a conventional 6-bp spacer (N6) and (ii) those with a potential 8-bp spacer (N8) [Barber and Zhurkin, J. Biomol. Struct. Dyn. 8 (1990) 213-232]. In this paper, we present the first experimental evidence that CRP binds to DNA with an N8 spacer with relatively high affinity, as measured by gel electrophoresis of CRP-DNA complexes. We have tested two types of N8 spacers: A+T-rich and G+C-rich. Compared with the affinity of CRP for a reference site with an N6 spacer, the binding strength of CRP toward an A+T-rich N8 sequence is lower and that toward a G+C-rich N8 site is comparable. Just like DNA sites with N6 spacers, those with N8 spacers utilize both halves of the symmetrical protein recognition sequences, TGTGA and TCACA. Because of the increased number of nucleotides in the N8 spacer, the two recognition sequences in DNA will have an increased distance and a helical twist between them. These would cause displacement of the two recognition sequences with respect to the two symmetrically located alpha-helices of the CRP dimer, if there is no change in the DNA conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Influence of fluctuations on DNA curvature. A comparison of flexible and static wedge models of intrinsically bent DNA.

Matrix-generator and Monte Carlo methods have been employed to study the influence of thermal fluctuations on the overall sizes and shapes of curved pieces of DNA. The DNA model involves the independent angular parameters relating successive base-pair steps: the sequence-dependent equilibrium values and fluctuations of the twist, tilt, and roll angles. The curved sequence under study is the (A5X5)n repeating polymer, the AA and XX steps having different equilibrium roll and twist values. Both planar circles and superhelices are analysed. Detailed comparison is made between the rigorous statistical mechanical representation of the DNA and simplified static models currently used in the literature. That is, a more realistic "flexible wedge" model is contrasted with the existing "static wedge" model of DNA curvature, which is demonstrated to be inadequate. The size of the coils is described by the unperturbed root-mean-square end-to-end distance and the shape by a ratio of the principal moments of the radius of gyration. The moment ratios indicate that when DNA is relatively short (e.g. its length is shorter than half a turn of the static superhelix), the flexible chains are more "short and thick" than the static structure. The end-to-end distances, however, are practically the same in the two models. For longer DNA fragments, the flexible chain is more extended in terms of the end-to-end distance and more globular in terms of the moment ratio. Thus, fluctuations "blur" the curvature of longer DNA fragments compared with static models. Furthermore, the overall average shape of slightly curved DNA subject to natural bending and twisting fluctuations is essentially indistinguishable from that of the corresponding "straight" DNA. Such configurational similarities are apparently responsible for the relative insensitivity of the polyacrylamide gel matrix to small degrees of DNA curvature. These findings raise serious questions regarding the quantitative estimation of wedge angles in DNA from electrophoretic experiments, based on static models. Comparison between planar circles and superhelices shows that when fluctuations are considered, the flexible circles are more spherical than the superhelices. The results imply that when DNA bending is exactly "in phase" with the helical repeat (i.e. when the DNA loop is exactly planar at 0 K), the DNA coil is packed more tightly than when bending and twisting are "out of phase" (and a superhelix is formed at 0 K). This finding is consistent with polyacrylamide gel electrophoresis data testifying to an increase in DNA retardation when twisting is more precisely "tuned".(ABSTRACT TRUNCATED AT 400 WORDS)

DNA↗