Search PubMed⌕ Search

Biomedical subjects

P T Tran

Publications and source records attributed to P T Tran.

15 recordsLinked to original sources
PubMed2001

MSH-MLH complexes formed at a DNA mismatch are disrupted by the PCNA sliding clamp.

In the yeast Saccharomyces cerevisiae, mismatch repair (MMR) is initiated by the binding of heterodimeric MutS homolog (MSH) complexes to mismatches that include single nucleotide and loop insertion/deletion mispairs. In in vitro experiments, the mismatch binding specificity of the MSH2-MSH6 heterodimer is eliminated if ATP is present. However, addition of the MutL homolog complex MLH1-PMS1 to binding reactions containing MSH2-MSH6, ATP, and mismatched substrate results in the formation of a stable ternary complex. The stability of this complex suggests that it represents an intermediate in MMR that is subsequently acted upon by other MMR factors. In support of this idea, we found that the replication processivity factor proliferating cell nuclear antigen (PCNA), which plays a critical role in MMR at step(s) prior to DNA resynthesis, disrupted preformed ternary complexes. These observations, in conjunction with experiments performed with streptavidin end-blocked mismatch substrates, suggested that PCNA interacts with an MSH-MLH complex formed on DNA mispairs.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Dissociable Rpb4-Rpb7 subassembly of rna polymerase II binds to single-strand nucleic acid and mediates a post-recruitment step in transcription initiation.

The Rpb4 and Rpb7 subunits of yeast RNA polymerase II form a heterodimeric complex essential for promoter-directed transcription initiation in a reconstituted system. Results of template competition experiments indicate that the Rpb4-Rpb7 complex is not required for stable recruitment of polymerase to active preinitiation complexes, suggesting that Rpb4-Rpb7 mediates an essential step subsequent to promoter binding. Sequence and structure-based alignments revealed a possible OB-fold single-strand nucleic acid-binding motif in Rpb7. Purified Rpb4-Rpb7 complex exhibited both single-strand DNA- and RNA-binding activities, and a small deletion in the putative OB-fold nucleic acid-binding surface of Rpb7 abolished binding activity without affecting the stability of the Rpb4-Rpb7 complex or its ability to associate with polymerase. The same mutation destroyed the transcription activity of the Rpb4-Rpb7 complex. A separate deletion elsewhere in the OB-fold motif of Rpb7 also blocked transcription but did not affect nucleic acid binding, suggesting that the OB-fold of Rpb7 mediates both DNA-protein and protein-protein interactions required for productive initiation.

Amino Acid Motifs↗
PubMed2000

Analysis of yeast MSH2-MSH6 suggests that the initiation of mismatch repair can be separated into discrete steps.

The yeast MSH2-MSH6 complex is required to repair both base-pair and single base insertion/deletion mismatches. MSH2-MSH6 binds to mismatch substrates and displays an ATPase activity that is modulated by mispairs that are repaired in vivo. To understand early steps in mismatch repair, we analyzed mismatch repair (MMR) defective MSH2-msh6-F337A and MSH2-msh6-340 complexes that contained amino acid substitutions in the MSH6 mismatch recognition domain. While both heterodimers were defective in forming stable complexes with mismatch substrates, only MSH2-msh6-340 bound to homoduplex DNA with an affinity that was similar to that observed for MSH2-MSH6. Additional analyses suggested that stable binding to a mispair is not sufficient to initiate recruitment of downstream repair factors. Previously, we observed that MSH2-MSH6 forms a stable complex with a palindromic insertion mismatch that escapes correction by MMR in vivo. Here we show that this binding is not accompanied by either a modulation in MSH2-MSH6 ATPase activity or an ATP-dependent recruitment of the MLH1-PMS1 complex. Together, these observations suggest that early stages in MMR can be divided into distinct recognition, stable binding, and downstream factor recruitment steps.

Adenosine Triphosphatases↗
PubMed2000

Tracking differential interference contrast diffraction line images with nanometre sensitivity.

This paper presents a computer vision framework for detecting and tracking diffraction images of linear structures in differential interference contrast (DIC) microscopy. The tracker can resolve image displacements of 1/10 of a pixel despite the weak and orientation-dependent contrast in DIC, as well as the variable blur in such image data caused by vertical specimen movement. In our high numerical aperture, high magnification microscope set-up, this resolution corresponds to 5 nm in object space. In video DIC similar resolution has been reported hitherto only for rotationally symmetric targets such as bead images. The tracker was developed for measuring deflections of clamped microtubules with a freely moving second end. By analysing the thermal fluctuations of such microtubules it was possible to derive their elasticity. The paper describes a filtering scheme for the detection and localization of DIC diffraction line images which represent loci of microtubules. For tracking the movements of the extracted lines we adopted the sum of squared (brightness) differences algorithm from computer vision. The analysis of the fluctuation measurements demonstrates the high sensitivity of this tracking technique in quantifying positional and orientational changes. We derived that the theoretical limit in tracking displacements of such diffraction line images is 1.25 nm, four times below the experimentally verified sensitivity. This indicates that the proposed tracker is still suboptimal. Nevertheless, the tracking precision was sufficient to reveal subtle deviations in the distribution of microtubule deflection from free diffusion. They were induced by pivotal points and multiple positions of relaxation. Also, the results suggest that there were defects in the polymer structure which caused very small but significant bends in the microtubule axis.

Algorithms↗
PubMed2000

Functional studies on the candidate ATPase domains of Saccharomyces cerevisiae MutLalpha.

Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLalpha, that is required for DNA mismatch repair after mismatch binding by MutS homologues. Recent sequence and structural studies have placed the NH(2) termini of MutL homologues in a new family of ATPases. To address the functional significance of this putative ATPase activity in MutLalpha, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo. Limited proteolysis with purified recombinant MutLalpha demonstrated that the NH(2) terminus of MutLalpha undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH(2) termini of Mlh1p and Pms1p. Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken together, these results suggest that MutLalpha undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Birefringence of single and bundled microtubules.

We have measured the birefringence of microtubules (MTs) and of MT-based macromolecular assemblies in vitro and in living cells by using the new Pol-Scope. A single microtubule in aqueous suspension and imaged with a numerical aperture of 1.4 had a peak retardance of 0.07 nm. The peak retardance of a small bundle increased linearly with the number of MTs in the bundle. Axonemes (prepared from sea urchin sperm) had a peak retardance 20 times higher than that of single MTs, in accordance with the nine doublets and two singlets arrangement of parallel MTs in the axoneme. Measured filament retardance decreased when the filament was defocused or the numerical aperture of the imaging system was decreased. However, the retardance "area," which we defined as the image retardance integrated along a line perpendicular to the filament axis, proved to be independent of focus and of numerical aperture. These results are in good agreement with a theory that we developed for measuring retardances with imaging optics. Our theoretical concept is based on Wiener's theory of mixed dielectrics, which is well established for nonimaging applications. We extend its use to imaging systems by considering the coherence region defined by the optical set-up. Light scattered from within that region interferes coherently in the image point. The presence of a filament in the coherence region leads to a polarization dependent scattering cross section and to a finite retardance measured in the image point. Similar to resolution measurements, the linear dimension of the coherence region for retardance measurements is on the order lambda/(2 NA), where lambda is the wavelength of light and NA is the numerical aperture of the illumination and imaging lenses.

Animals↗
PubMed1997

A metastable intermediate state of microtubule dynamic instability that differs significantly between plus and minus ends.

The current two-state GTP cap model of microtubule dynamic instability proposes that a terminal crown of GTP-tubulin stabilizes the microtubule lattice and promotes elongation while loss of this GTP-tubulin cap converts the microtubule end to shortening. However, when this model was directly tested by using a UV microbeam to sever axoneme-nucleated microtubules and thereby remove the microtubule's GTP cap, severed plus ends rapidly shortened, but severed minus ends immediately resumed elongation (Walker, R.A., S. Inoué, and E.D. Salmon. 1989. J. Cell Biol. 108: 931-937). To determine if these previous results were dependent on the use of axonemes as seeds or were due to UV damage, or if they instead indicate an intermediate state in cap dynamics, we performed UV cutting of self-assembled microtubules and mechanical cutting of axoneme-nucleated microtubules. These independent methods yielded results consistent with the original work: a significant percentage of severed minus ends are stable after cutting. In additional experiments, we found that the stability of both severed plus and minus ends could be increased by increasing the free tubulin concentration, the solution GTP concentration, or by assembling microtubules with guanylyl-(alpha,beta)-methylene-diphosphonate (GMPCPP). Our results show that stability of severed ends, particularly minus ends, is not an artifact, but instead reveals the existence of a metastable kinetic intermediate state between the elongation and shortening states of dynamic instability. The kinetic properties of this intermediate state differ between plus and minus ends. We propose a three-state conformational cap model of dynamic instability, which has three structural states and four transition rate constants, and which uses the asymmetry of the tubulin heterodimer to explain many of the differences in dynamic instability at plus and minus ends.

Animals↗
PubMed1997

Molecular cloning of the human methylthioadenosine phosphorylase processed pseudogene and localization to 3q28.

Human methylthioadenosine phosphorylase (MTAP) is a purine and methionine metabolic enzyme present ubiquitously in all normal tissues, but often deleted in many types of cancer. The gene for this enzyme maps to chromosome 9 at band p21 where the cyclin-dependent kinase inhibitor genes for p16 and p15 also reside. During our efforts to clone this gene we also isolated a phage clone containing a processed pseudogene of MTAP. The sequence is 92% homologous to the MTAP cDNA, is flanked at its 3' end by a repetitive element, but does not possess a poly(A) stretch. We localized this processed pseudogene to band 28 on the long arm of chromosome 3 by fluorescence in situ hybridization. All 22 malignant cell lines with deletions at 9p21 screened possessed the pseudogene.

Amino Acid Sequence↗
PubMed1997

How tubulin subunits are lost from the shortening ends of microtubules.

Microtubules exhibit dynamic instability, switching between persistent states of growth and shortening at their ends. The switch between growth and shortening has been proposed to depend on end conformation where growing ends have "straight" tubulin protofilaments stabilized by a terminal cap of GTP-tubulin, while-shortening ends have lost their GTP-tubulin cap, allowing terminal GDP-tubulin dimers to curve inside-out and peel rapidly away from the microtubule lattice. This "conformational cap" model predicts that tubulin dissociation from shortening ends is a two-step process where the average lengths of curved GDP-tubulin protofilaments at a depolymerizing end will depend on the ratio of the rate of peeling to the rate of breakage of the longitudinal bonds between adjacent curved dimers. We have tested this model for the plus and minus ends of microtubules assembled with pure porcine tubulin off the ends of axoneme fragments in standard assembly buffer. Individual microtubule ends were imaged using video-enhanced differential interference contrast light microscopy. The rate of rapid shortening was systematically increased by isothermal dilution into assembly buffer containing various concentrations of Mg2+ or Ca2+ ions. At 1 mM Mg2+ and no Ca2+, shortening occurred at 20 (plus) and 45 (minus) microns/min. The ends appeared similar in contrast to growing ends and the core of the microtubule and the ends appeared blunt or slightly frayed by negative stain electron microscopy. Above 20 mM Mg2+ or above 5 mM Ca2+, microtubule shortening occurred at 60 (plus) and 115 (minus) microns/min or faster and "knobs" were distinctly visible at depolymerizing ends, particularly at the faster minus ends, and knob contrast remained constant during many micrometers of rapid shortening. Negative stain electron microscopy revealed that these knobs were "blossoms" of inside-out curved protofilaments, some extending for several helical turns (30 to 60 dimers in length) at constant curvature from the ends. At these high shortening velocities, the peeling of curved protofilaments was confined to within several dimers of the end of the microtubule cylinder, suggesting that dimer curling and protofilament peeling is constrained to the tip by interactions between adjacent straight protofilaments. Depolymerization is produced by conformational changes in GDP-tubulin since microtubules assembled with a slowly hydrolizable analog of GTP, GMPCPP, are stable even at 20 mM Mg2+ or 5 mM Ca2+. Monte Carlo simulations show that the ratio of the peeling to breakage rate constants can control the steady-state average length of curved GDP-tubulin protofilaments at the depolymerizing end.

Animals↗
PubMed1996

Routine neonatal circumcision?

Routine neonatal circumcision is still a controversial procedure. This article attempts to clarify some of the advantages and disadvantages of neonatal circumcision. The increased rate of penile cancer among uncircumcised men appears to justify the procedure, but that alone is not sufficient justification. The final decision on neonatal circumcision should be made by parents with balanced counsel from attending physicians.

Circumcision, Male↗