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Dynamics and function of compact nucleosome arrays.

Poirier MG, Oh E, Tims HS, Widom J - Nat. Struct. Mol. Biol. (2009)

Bottom Line: Compact states of the arrays allow binding to DNA within the central nucleosome via site exposure.Protein binding can also drive decompaction of the arrays.Thus, our results reveal multiple modes by which spontaneous chromatin fiber dynamics allow for the invasion and action of DNA-processing protein complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois, USA.

ABSTRACT
The packaging of eukaryotic DNA into chromatin sterically occludes polymerases, recombinases and repair enzymes. How chromatin structure changes to allow their actions is unknown. We constructed defined fluorescently labeled trinucleosome arrays, allowing analysis of chromatin conformational dynamics via fluorescence resonance energy transfer (FRET). The arrays undergo reversible Mg2+-dependent folding similar to that of longer arrays studied previously. We define two intermediate conformational states in the reversible folding of the nucleosome arrays and characterize the microscopic rate constants. Nucleosome arrays are highly dynamic even when compact, undergoing conformational fluctuations on timescales in the second to microsecond range. Compact states of the arrays allow binding to DNA within the central nucleosome via site exposure. Protein binding can also drive decompaction of the arrays. Thus, our results reveal multiple modes by which spontaneous chromatin fiber dynamics allow for the invasion and action of DNA-processing protein complexes.

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Minimal kinetic scheme for nucleosome array dynamics in 1 mM Mg2+The stopped-flow FRET experiments (Fig. 4d,f,g) exhibit biphasic compaction kinetics, requiring a minimum of three conformational states, with the second forward step (S3 → S2, rate k32) and reverse step (S2 → S3, rate k23) being slower than the first forward step (S4 → S3, rate k43). The equilibrium titrations (Fig. 1) demonstrate that the arrays are compact (in the time average) in 1 mM Mg2+; yet the FRET-FCS experiment (Fig. 4a,c) demonstrates rapid interconversion between two states, with a relaxation time of ~10-5 sec, much faster than the rates for S3 ←→ S2, therefore requiring at least one additional compact state (S1) connected reversibly to S2. The corresponding rates (or the bounds on them) imply that only states S1 and S2 are significantly populated in 1 mM Mg2+; yet the central nucleosomes of compact arrays undergo nucleosomal site exposure and bind LexA protein, just as for mononucleosomes. Thus states S1 and/or S2 are competent for nucleosomal site exposure. See Supplementary text for further details of the kinetic analysis. The structures shown are intended only to represent that compactness increases progressively (decreasing distance from nucleosome 1 to nucleosome 3) as the arrays evolve from state S4 → S1.
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Figure 5: Minimal kinetic scheme for nucleosome array dynamics in 1 mM Mg2+The stopped-flow FRET experiments (Fig. 4d,f,g) exhibit biphasic compaction kinetics, requiring a minimum of three conformational states, with the second forward step (S3 → S2, rate k32) and reverse step (S2 → S3, rate k23) being slower than the first forward step (S4 → S3, rate k43). The equilibrium titrations (Fig. 1) demonstrate that the arrays are compact (in the time average) in 1 mM Mg2+; yet the FRET-FCS experiment (Fig. 4a,c) demonstrates rapid interconversion between two states, with a relaxation time of ~10-5 sec, much faster than the rates for S3 ←→ S2, therefore requiring at least one additional compact state (S1) connected reversibly to S2. The corresponding rates (or the bounds on them) imply that only states S1 and S2 are significantly populated in 1 mM Mg2+; yet the central nucleosomes of compact arrays undergo nucleosomal site exposure and bind LexA protein, just as for mononucleosomes. Thus states S1 and/or S2 are competent for nucleosomal site exposure. See Supplementary text for further details of the kinetic analysis. The structures shown are intended only to represent that compactness increases progressively (decreasing distance from nucleosome 1 to nucleosome 3) as the arrays evolve from state S4 → S1.

Mentions: The simplest kinetic mechanism that integrates the equilibrium and kinetic data for 1 mM Mg2+ (Figs.1 and 4, respectively) requires a minimum of four conformational states (i.e., states with differing FRET efficiencies) (Fig. 5, and Supplemental text), with at least two conformational intermediates between the most-compact and most-extended states of the arrays.


Dynamics and function of compact nucleosome arrays.

Poirier MG, Oh E, Tims HS, Widom J - Nat. Struct. Mol. Biol. (2009)

Minimal kinetic scheme for nucleosome array dynamics in 1 mM Mg2+The stopped-flow FRET experiments (Fig. 4d,f,g) exhibit biphasic compaction kinetics, requiring a minimum of three conformational states, with the second forward step (S3 → S2, rate k32) and reverse step (S2 → S3, rate k23) being slower than the first forward step (S4 → S3, rate k43). The equilibrium titrations (Fig. 1) demonstrate that the arrays are compact (in the time average) in 1 mM Mg2+; yet the FRET-FCS experiment (Fig. 4a,c) demonstrates rapid interconversion between two states, with a relaxation time of ~10-5 sec, much faster than the rates for S3 ←→ S2, therefore requiring at least one additional compact state (S1) connected reversibly to S2. The corresponding rates (or the bounds on them) imply that only states S1 and S2 are significantly populated in 1 mM Mg2+; yet the central nucleosomes of compact arrays undergo nucleosomal site exposure and bind LexA protein, just as for mononucleosomes. Thus states S1 and/or S2 are competent for nucleosomal site exposure. See Supplementary text for further details of the kinetic analysis. The structures shown are intended only to represent that compactness increases progressively (decreasing distance from nucleosome 1 to nucleosome 3) as the arrays evolve from state S4 → S1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2748796&req=5

Figure 5: Minimal kinetic scheme for nucleosome array dynamics in 1 mM Mg2+The stopped-flow FRET experiments (Fig. 4d,f,g) exhibit biphasic compaction kinetics, requiring a minimum of three conformational states, with the second forward step (S3 → S2, rate k32) and reverse step (S2 → S3, rate k23) being slower than the first forward step (S4 → S3, rate k43). The equilibrium titrations (Fig. 1) demonstrate that the arrays are compact (in the time average) in 1 mM Mg2+; yet the FRET-FCS experiment (Fig. 4a,c) demonstrates rapid interconversion between two states, with a relaxation time of ~10-5 sec, much faster than the rates for S3 ←→ S2, therefore requiring at least one additional compact state (S1) connected reversibly to S2. The corresponding rates (or the bounds on them) imply that only states S1 and S2 are significantly populated in 1 mM Mg2+; yet the central nucleosomes of compact arrays undergo nucleosomal site exposure and bind LexA protein, just as for mononucleosomes. Thus states S1 and/or S2 are competent for nucleosomal site exposure. See Supplementary text for further details of the kinetic analysis. The structures shown are intended only to represent that compactness increases progressively (decreasing distance from nucleosome 1 to nucleosome 3) as the arrays evolve from state S4 → S1.
Mentions: The simplest kinetic mechanism that integrates the equilibrium and kinetic data for 1 mM Mg2+ (Figs.1 and 4, respectively) requires a minimum of four conformational states (i.e., states with differing FRET efficiencies) (Fig. 5, and Supplemental text), with at least two conformational intermediates between the most-compact and most-extended states of the arrays.

Bottom Line: Compact states of the arrays allow binding to DNA within the central nucleosome via site exposure.Protein binding can also drive decompaction of the arrays.Thus, our results reveal multiple modes by which spontaneous chromatin fiber dynamics allow for the invasion and action of DNA-processing protein complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois, USA.

ABSTRACT
The packaging of eukaryotic DNA into chromatin sterically occludes polymerases, recombinases and repair enzymes. How chromatin structure changes to allow their actions is unknown. We constructed defined fluorescently labeled trinucleosome arrays, allowing analysis of chromatin conformational dynamics via fluorescence resonance energy transfer (FRET). The arrays undergo reversible Mg2+-dependent folding similar to that of longer arrays studied previously. We define two intermediate conformational states in the reversible folding of the nucleosome arrays and characterize the microscopic rate constants. Nucleosome arrays are highly dynamic even when compact, undergoing conformational fluctuations on timescales in the second to microsecond range. Compact states of the arrays allow binding to DNA within the central nucleosome via site exposure. Protein binding can also drive decompaction of the arrays. Thus, our results reveal multiple modes by which spontaneous chromatin fiber dynamics allow for the invasion and action of DNA-processing protein complexes.

Show MeSH
Related in: MedlinePlus