15 research outputs found
Elucidation of the Mechanisms of Nucleosome Binding and Repositioning by a Chromatin Remodeler: Monomeric ISWI Remodels Nucleosomes Through a Random Walk
The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The goal of this work was to characterize quantitatively the nucleosome repositioning activity, and associated processes of nucleotide binding, DNA binding, and nucleosome binding, of the chromatin remodeler ISWI. ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. The necessary first step in determining how these essential enzymes catalyze the repositioning of nucleosomes is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. Toward this goal we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon ISWI binding to these substrates. We determined that one ISWI molecule binds to a 20 bp double stranded DNA substrate with an affinity of (18 ± 2) nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants 1/&beta1 = (1.3 ± 0.6) nM and 1/&beta2 = (13 ± 7) nM2. Furthermore, in order to better understand the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogs on substrate binding by ISWI. While the affinity of ISWI to binding nucleosome substrate with short lengths of flanking DNA was not affected by presence of nucleotides, the affinity of ISWI for binding DNA substrate is weakened in the presence of non-hydrolysable ATP analogs but not in the presence of ADP. Additionally, using standard electrophoresis assays we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing different lengths of DNA symmetrically flanking an initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a novel spectrophotometric assay for nucleosome repositioning we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed under various ISWI concentrations revealed that monomeric ISWI is sufficient to account for the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning suggesting that DNA translocation by ISWI is not energetically rate limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction. In conclusion, the characterization of the mechanism of nucleosome binding and repositioning by the chromatin remodeler ISWI presented in this dissertation provides a foundation for future studies aiming to understand how various regulatory elements influence the function of ISWI
Effects of nucleosome stability on remodeler-catalyzed repositioning
Chromatin remodelers are molecular motors that play essential roles in the regulation of nucleosome positioning and chromatin accessibility. These machines couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of manipulating chromatin structure through processes that are not completely understood. Here we present a quantitative analysis of nucleosome repositioning by the imitation switch (ISWI) chromatin remodeler and demonstrate that nucleosome stability significantly impacts the observed activity. We show how DNA damage induced changes in the affinity of DNA wrapping within the nucleosome can affect ISWI repositioning activity and demonstrate how assay-dependent limitations can bias studies of nucleosome repositioning. Together, these results also suggest that some of the diversity seen in chromatin remodeler activity can be attributed to the variations in the thermodynamics of interactions between the remodeler, the histones, and the DNA, rather than reflect inherent properties of the remodeler itself
The N-Terminal Domain and Glycosomal Localization of Leishmania Initial Acyltransferase LmDAT Are Important for Lipophosphoglycan Synthesis
Ether glycerolipids of Leishmania major are important membrane components as well as building blocks of various virulence factors. In L. major, the first enzyme of the ether glycerolipid biosynthetic pathway, LmDAT, is an unusual, glycosomal dihydroxyacetonephosphate acyltransferase important for parasite's growth and survival during the stationary phase, synthesis of ether lipids, and virulence. The present work extends our knowledge of this important biosynthetic enzyme in parasite biology. Site-directed mutagenesis of LmDAT demonstrated that an active enzyme was critical for normal growth and survival during the stationary phase. Deletion analyses showed that the large N-terminal extension of this initial acyltransferase may be important for its stability or activity. Further, abrogation of the C-terminal glycosomal targeting signal sequence of LmDAT led to extraglycosomal localization, did not impair its enzymatic activity but affected synthesis of the ether glycerolipid-based virulence factor lipophosphoglycan. In addition, expression of this recombinant form of LmDAT in a null mutant of LmDAT did not restore normal growth and survival during the stationary phase. These results emphasize the importance of this enzyme's compartmentalization in the glycosome for the generation of lipophosphoglycan and parasite's biology
The role of the dihydroxyacetone phosphate acyltransferase LmDAT in lipophosphoglycan synthesis, metacyclogenesis and autophagy in Leishmania major
Master of ScienceDepartment of BiochemistryRachel ZuffereyGlycerolipids are the most abundant lipids and are important constituents of various virulence factors in the protozoan parasite Leishmania. The dihydroxyacetone phosphate acyltransferase LmDAT catalyzes the first step of the ether, and possibly ester glycerolipid biosynthetic pathway. A L. major null mutant of LmDAT grew slowly, died rapidly during the stationary phase of growth, and more importantly, was attenuated in virulence in mice. The goal of this study was to determine the molecular basis responsible for the attenuated virulence. Western blot analysis revealed that the ∆lmdat/∆lmdat null mutant synthesized altered versions of the virulence factor lipophosphoglycans that were not released in the media, suggesting that its lipid anchor structure was altered. The ∆lmdat/∆lmdat strain differentiated into virulent metacyclics, but with lower efficiency compared to the wild type. Using the autophagosomal marker ATG8-GFP, the ∆lmdat/∆lmdat line produced twice as many autophagosomes as the wild type, suggesting that it is either defective in degradation of autophagosomes or that autophagy is simply induced. In conclusion, the attenuated virulence of ∆lmdat/∆lmdat may be explained by i) its inability to synthesize and release normal forms of lipophosphoglycan, ii) its inability to fully differentiate into virulent metacyclics, and iii) altered autophagy
ISWI Remodels Nucleosomes through a Random Walk
The chromatin remodeler ISWI is capable
of repositioning clusters
of nucleosomes to create well-ordered arrays or moving single nucleosomes
from the center of DNA fragments toward the ends without disrupting
their integrity. Using standard electrophoresis assays, we have monitored
the ISWI-catalyzed repositioning of different nucleosome samples each
containing a different length of DNA symmetrically flanking the initially
centrally positioned histone octamer. We find that ISWI moves the
histone octamer between distinct and thermodynamically stable positions
on the DNA according to a random walk mechanism. Through the application
of a spectrophotometric assay for nucleosome repositioning, we further
characterized the repositioning activity of ISWI using short nucleosome
substrates and were able to determine the macroscopic rate of nucleosome
repositioning by ISWI. Additionally, quantitative analysis of repositioning
experiments performed at various ISWI concentrations revealed that
a monomeric ISWI is sufficient to obtain the observed repositioning
activity as the presence of a second ISWI bound had no effect on the
rate of nucleosome repositioning. We also found that ATP hydrolysis
is poorly coupled to nucleosome repositioning, suggesting that DNA
translocation by ISWI is not energetically rate-limiting for the repositioning
reaction. This is the first calculation of a microscopic ATPase coupling
efficiency for nucleosome repositioning and also further supports
our conclusion that a second bound ISWI does not contribute to the
repositioning reaction
Characterization of mutant forms of <i>Lm</i>DAT.
<p>(A) Schematic representation of human DHAPAT (<i>h</i>DHAPAT) and mutant forms of <i>Lm</i>DAT. The grey rectangle, the black rectangle and the hatched area depict the HV tag, the conserved domain, and the C-terminal glycosomal targeting tripeptide, respectively, and the asterisk depicts the point mutation. B) DHAPAT activity was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027802#s2" target="_blank">Materials and Methods</a>. Equivalent of 0.5 mg protein extracts were applied for the assay. Null mutant alone or expressing HV-tagged wild-type and mutant forms of <i>Lm</i>DAT were used as a source of protein extracts. Activity is expressed as percentage of the positive control, the wild type (WT). The assay was performed twice in duplicate, and the graph depicts one representative experiment. Standard deviations are shown. (C) Western blot analyses in the presence of V5-specific (upper; V5) and hypoxanthine guanine phosphoribosyltransferase specific (lower; HGPRT; loading control) antibodies. Equivalent of 5×10<sup>7</sup> cells were loaded in each lane. The apparent molecular weight is shown on the left. (D) Western blot analysis in the presence of WIC79.3 antibody to detect LPG. Equivalent of 10<sup>6</sup> cells were loaded in each lane. (B, C, D): 1, <i>Δlmdat/Δlmdat</i>; 2, <i>Δlmdat/Δlmdat [HV-LmDAT NEO]</i>; 3, <i>Δlmdat/Δlmdat [HV-ΔN<sub>546</sub>-LmDAT NEO]</i>; 4, <i>Δlmdat/Δlmdat [HV-ΔN<sub>686</sub>-LmDAT NEO]</i>; 5, <i>Δlmdat/Δlmdat [HV-LmDAT-ΔC<sub>733</sub> NEO]</i>; 6, <i>Δlmdat/Δlmdat [HV-LmDAT-ΔC<sub>3</sub> NEO]</i>; 7, <i>Δlmdat/Δlmdat [HV-LmDAT<sup>K852L</sup> NEO]</i>; WT, wild type.</p
HV-<i>Lm</i>DAT-ΔC<sub>3</sub> does not localize in the glycosomes.
<p>Wild type expressing recombinant HV-<i>Lm</i>DAT-ΔC<sub>3</sub> was analyzed by phase contrast (panel 1) or immunofluorescence microscopy using anti-V5 antibody (panel 2) or polyclonal antiserum specific to hypoxanthine guanine phosphoribosyltransferase (panel 3). Panel 4 shows the merge of panels 2 and 3.</p
Glycerolipid biosynthetic pathways in <i>Leishmania</i>.
<p>AGAT, 1-acyl-glycerol-3-phosphate acyltransferase; ADR, alkyl/acyl-DHAP reductase; <i>Lm</i>ADS, alkyl-DHAP synthase; DHAP, dihydroxyacetonephosphate; <i>Lm</i>DAT, DHAP acyltransferase; <i>Lm</i>FAR, fatty acyl-CoA reductase; G3P, glycerol-3-phosphate; <i>Lm</i>GAT, G3P acyltransferase; PA, phosphatidic acid. Genes encoding ADR and AGAT in <i>Leishmania</i> are unknown.</p
Growth curves.
<p>Cells were inoculated at a cell density of 5×10<sup>5</sup>/ml and were enumerated with a hemacytometer as a function of time. The assay was performed twice and the graphs represent a typical experiment. Standard deviations are shown. (A) Black circles, wild type; grey circles, complemented line <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT NEO]</i>; white circles, <i>Δlmdat</i>/<i>Δlmdat</i>; white triangles, <i>Δlmdat/Δlmdat [HV-ΔN<sub>546</sub>-LmDAT NEO]</i>; grey triangles, <i>Δlmdat/Δlmdat [HV-ΔN<sub>686</sub>-LmDAT NEO]</i>; black triangles, <i>Δlmdat/Δlmdat [HV-LmDAT-ΔC<sub>733</sub> NEO]</i>. (B) Black circles, wild type; grey circles, complemented line <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT NEO]</i>; white circles, <i>Δlmdat</i>/<i>Δlmdat</i>; white triangles, <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT-ΔC<sub>3</sub> NEO]</i>; grey triangles, <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT<sup>K852L</sup> NEO]</i>.</p