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P Shing Ho

Publications and source records attributed to P Shing Ho.

12 recordsLinked to original sources
PubMed2006

Solution formation of Holliday junctions in inverted-repeat DNA sequences.

The structure of Holliday junctions has now been well characterized at the atomic level through single-crystal X-ray diffraction in symmetric (inverted-repeat) DNA sequences. At issue, however, is whether the formation of these four-stranded complexes in solution is truly sequence dependent in the manner proposed or is an artifact of the crystallization process and, therefore, has no relevance to the behavior of this central intermediate in homologous recombination and recombination-dependent cellular processes. Here, we apply analytical ultracentrifugation to demonstrate that the sequence d(CCGGTACCGG), which crystallizes in the stacked-X form of the junction, assembles into four-stranded junctions in solution in a manner that is dependent on the DNA and cation concentrations, with an equilibrium established between the junction and duplex forms at 100-200 microM DNA duplex. In contrast, the sequence d(CCGCTAGCGG), which has been crystallized as B-DNA, is seen to adopt only the double-helical form at all DNA and salt concentrations that were tested. Thus, the ACC trinucleotide core is now shown to be important for the formation of Holliday junctions in both crystals and in solution and can be estimated to contribute approximately -4 kcal/mol to stabilizing this recombination intermediate in inverted-repeat sequences.

Crystallography, X-Ray↗
PubMed2005

How sequence defines structure: a crystallographic map of DNA structure and conformation.

The fundamental question of how sequence defines conformation is explicitly answered if the structures of all possible sequences of a macromolecule are determined. We present here a crystallographic screen of all permutations of the inverted repeat DNA sequence d(CCnnnN6N7N8GG), where N6, N7, and N8 are any of the four naturally occurring nucleotides. At this point, 63 of the 64 possible permutations have been crystallized from a defined set of solutions. When combined with previous work, we have assembled a data set of 37 single-crystal structures from 29 of the sequences in this motif, representing three structural classes of DNA (B-DNA, A-DNA, and four-stranded Holliday junctions). This data set includes a unique set of amphimorphic sequence, those that crystallize in two different conformations and serve to bridge the three structural phases. We have thus constructed a map of DNA structures that can be walked through in single nucleotide steps. Finally, the resulting data set allows us to dissect in detail the stabilization of and conformational variations within structural classes and identify significant conformational deviations within a particular structural class that result from sequence rather than crystal or crystallization effects.

Base Sequence↗
PubMed2005

Topolins and hydroxylated thidiazuron derivatives are substrates of cytokinin O-glucosyltransferase with position specificity related to receptor recognition.

Glucosides of trans-zeatin occur widely in plant tissues, formed either by O-glucosylation of the hydroxylated side chain or N-glucosylation of the purine ring structure. O-Glucosylation is stereo-specific: the O-glucosyltransferase encoded by the Phaseolus lunatus ZOG1 gene has high affinity for trans-zeatin as the substrate, whereas the enzyme encoded by the maize (Zea mays) cisZOG1 gene prefers cis-zeatin. Here we show that hydroxylated derivatives of benzyladenine (topolins) are also substrates of ZOG1 and cisZOG1. The m-OH and o-OH derivatives are the preferred substrate of ZOG1 and cisZOG1, respectively. Among the hydroxylated derivatives of thidiazuron tested, the only enzyme/substrate combination resulting in conversion was cisZOG1/(o-OH) thidiazuron. The abilities of these cytokinins to serve as substrates to the glucosyltransferases were in a large part correlated with their biological activities in the P. lunatus callus bioassay, indicating that there may be similarities between cytokinin-binding sites on the enzymes and cytokinin receptors. Further support for this interpretation is provided by cytokinin recognition studies involving the Arabidopsis (Arabidopsis thaliana) CRE1/WOL/AHK4 and maize ZmHK1 receptors. The AHK4 receptor responded to trans-zeatin and m-topolin, while the ZmHK1 receptor responded also to cis-zeatin and o-topolin. Three-dimensional molecular models of the substrates were applied to explain the results.

Adenine↗
PubMed2004

Distributions of Z-DNA and nuclear factor I in human chromosome 22: a model for coupled transcriptional regulation.

An analysis of the human chromosome 22 genomic sequence shows that both Z-DNA forming regions (ZDRs) and promoter sites for nuclear factor-I (NFI) are correlated with the locations of known and predicted genes across the chromosome and accumulate around the transcriptional start sites of the known genes. Thus, the occurrence of Z-DNA across human genomic sequences mirrors that of a known eukaryotic transcription factor. In addition, 43 of the 383 fully annotated chromosomal genes have ZDRs within 2 nucleosomes upstream of strong NFIs. This suggests a distinct class of human genes that may potentially be transcriptionally regulated by a mechanism that couples Z-DNA with NFI activation, similar to the mechanism previously elucidated for the human colony stimulation factor-I promoter [Liu et al. (2001) Cell, 106, 309-318]. The results from this study will facilitate the design of experimental studies to test the generality of this mechanism for other genes in the cell.

Binding Sites↗
PubMed2004

Halogen bonds in biological molecules.

Short oxygen-halogen interactions have been known in organic chemistry since the 1950s and recently have been exploited in the design of supramolecular assemblies. The present survey of protein and nucleic acid structures reveals similar halogen bonds as potentially stabilizing inter- and intramolecular interactions that can affect ligand binding and molecular folding. A halogen bond in biomolecules can be defined as a short C-X...O-Y interaction (C-X is a carbon-bonded chlorine, bromine, or iodine, and O-Y is a carbonyl, hydroxyl, charged carboxylate, or phosphate group), where the X...O distance is less than or equal to the sums of the respective van der Waals radii (3.27 A for Cl...O, 3.37 A for Br...O, and 3.50 A for I...O) and can conform to the geometry seen in small molecules, with the C-X...O angle approximately 165 degrees (consistent with a strong directional polarization of the halogen) and the X...O-Y angle approximately 120 degrees . Alternative geometries can be imposed by the more complex environment found in biomolecules, depending on which of the two types of donor systems are involved in the interaction: (i) the lone pair electrons of oxygen (and, to a lesser extent, nitrogen and sulfur) atoms or (ii) the delocalized pi -electrons of peptide bonds or carboxylate or amide groups. Thus, the specific geometry and diversity of the interacting partners of halogen bonds offer new and versatile tools for the design of ligands as drugs and materials in nanotechnology.

Cytosine↗
PubMed2004

Influence of minor groove substituents on the structure of DNA Holliday junctions.

The inosine-containing sequence d(CCIGTACm(5)CGG) is shown to crystallize as a four-stranded DNA junction. This structure is nearly identical to the antiparallel junction formed by the parent d(CCGGTACm(5)()CGG) sequence [Vargason, J. M., and Ho, P. S. (2002) J. Biol. Chem. 277, 21041-21049] in terms of its conformational geometry, and inter- and intramolecular interactions within the DNA and between the DNA and solvent, even though the 2-amino group in the minor groove of the important G(3).m(5)C(8) base pair of the junction core trinucleotide (italicized) has been removed. In contrast, the analogous 2,6-diaminopurine sequence d(CCDGTACTGG) crystallizes as resolved duplex DNAs, just like its parent sequence d(CCAGTACTGG) [Hays, F. A., Vargason, J. M., and Ho, P. S. (2003) Biochemistry 42, 9586-9597]. These results demonstrate that it is not the presence or absence of the 2-amino group in the minor groove of the R(3).Y(8) base pair that specifies whether a sequence forms a junction, but the positions of the extracyclic amino and keto groups in the major groove. Finally, the study shows that the arms of the junction can accommodate perturbations to the B-DNA conformation of the stacked duplex arms associated with the loss of the 2-amino substituent, and that two hydrogen bonding interactions from the C(7) and Y(8) pyrimidine nucleotides to phosphate oxygens of the junction crossover specify the geometry of the Holliday junction.

2-Aminopurine↗
PubMed2004

Definitions and analysis of DNA Holliday junction geometry.

A number of single-crystal structures have now been solved of the four-stranded antiparallel stacked-X form of the Holliday junction. These structures demonstrate how base sequence, substituents, and drug and ion interactions affect the general conformation of this recombination intermediate. The geometry of junctions had previously been described in terms of a specific set of parameters that include: (i) the angle relating the ends of DNA duplexes arms of the junction (interduplex angle); (ii) the relative rotation of the duplexes about the helix axes of the stacked duplex arms (J(roll)); and (iii) the translation of the duplexes along these helix axes (J(slide)). Here, we present a consistent set of definitions and methods to accurately calculate each of these parameters based on the helical features of the stacked duplex arms in the single-crystal structures of the stacked-X junction, and demonstrate how each of these parameters contributes to an overall conformational feature of the structure. We show that the values for these parameters derived from global rather than local helical axes through the stacked bases of the duplex arms are the most representative of the stacked-X junction conformation. In addition, a very specific parameter (J(twist)) is introduced which relates the relative orientation of the stacked duplex arms across the junction which, unlike the interduplex angle, is length independent. The results from this study provide a general means to relate the geometric features seen in the crystal structures to those determined in solution.

Base Sequence↗
PubMed2003

Mass spectrometric approaches using electrospray ionization charge states and hydrogen-deuterium exchange for determining protein structures and their conformational changes.

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.

Deuterium↗
PubMed2003

Effect of sequence on the conformation of DNA holliday junctions.

Structures of the DNA sequences d(CCGGCGCCGG) and d(CCAGTACbr(5)UGG) are presented here as four-way Holliday junctions in their compact stacked-X forms, with antiparallel alignment of the DNA strands. Thus, the ACC-trinucleotide motif, previously identified as important for stabilizing the junction, is now extended to PuCPy, where Pu is either an adenine or guanine, and Py is either a cytosine, 5-methylcytosine, or 5-bromouracil but not thymine nucleotide. We see that both sequence and base substituents affect the geometry of the junction in terms of the interduplex angle as well as the previously defined conformational variables, J(roll) (the rotation of the stacked duplexes about their respective helical axis) and J(slide) (the translational displacement of the stacked duplexes along their respective helical axis). The structures of the GCC and parent ACC containing junctions fall into a distinct conformational class that is relatively undistorted in terms of J(slide) and J(roll), with interduplex angles of 40-43 degrees. The substituted ACbr(5)U structure, however, is more akin to that of the distorted methylated ACm(5)C containing junction, with J(slide) (>or=2.3 A) and a similar J(roll) (164 degrees) opening the major groove-side of the junction, but shows a reduced interduplex angle. In contrast, the analogous d(CCAGTACTGG) sequence has to date been crystallized only as resolved B-DNA duplexes. This suggests that there is an electronic effect of substituents at the pyrimidine Py position on the stability of four-stranded junctions. The single-crystal structures presented here, therefore, show how sequence affects the detailed geometry, and subsequently, the associated stability and conformational dynamics of the Holliday junction.

Base Composition↗
PubMed2002

The effect of cytosine methylation on the structure and geometry of the Holliday junction: the structure of d(CCGGTACm5CGG) at 1.5 A resolution.

The single crystal structure of the methylated sequence d(CCGGTACm(5)CGG) has been solved as an antiparallel stacked X Holliday junction to 1.5 A resolution. When compared with the parent nonmethylated d(CCGGTACCGG) structure, the duplexes are translated by 3.4 A along the helix axis and rotated by 10.8 degrees relative to each other, rendering the major grooves more accessible overall. A Ca(2+) complex is seen in the minor groove opposite the junction but is related to the B conformation of the stacked arms. At the junction itself, the hydrogen bond from the N4 nitrogen of cytosine C8 to the C7 phosphate at the crossover in the parent structure has been replaced by a water bridge. Thus, this direct interaction is not absolutely required to stabilize the junction at the previously defined ACC trinucleotide core. The more compact methylated junction forces the Na(+) of the protected central cavity of the nonmethylated junction into a solvent cluster that spans the space between the junction crossover and the stacked arms. A series of void volumes within the methylated and the nonmethylated structures suggests that small monovalent cations can fill and vacate this central cavity without the need to unfold the four-stranded Holliday junction completely.

Base Sequence↗
PubMedDate not supplied

The stacked-X DNA Holliday junction and protein recognition.

The crystal structure of the four-stranded DNA Holliday junction has now been determined in the presence and absence of junction binding proteins, with the extended open-X form of the junction seen in all protein complexes, but the more compact stacked-X structure observed in free DNA. The structures of the stacked-X junction were crystallized because of an unexpected sequence dependence on the stability of this structure. Inverted repeat sequences that contain the general motif NCC or ANC favor formation of stacked-X junctions, with the junction cross-over occurring between the first two positions of the trinucleotides. This review focuses on the sequence dependent structure of the stacked-X junction and how it may play a role in structural recognition by a class of dimeric junction resolving enzymes that themselves show no direct sequence recognition.

Base Sequence↗