Compaction kinetics on single DNAs: purified nucleosome reconstitution systems versus crude extract

Abstract

Kinetics of compaction on single DNA molecules are studied by fluorescence videomicroscopy in the presence of 1), Xenopus egg extracts and 2), purified nucleosome reconstitution systems using a combination of histones with either the histone chaperone Nucleosome Assembly Protein (NAP-1) or negatively charged macromolecules such as polyglutamic acid and RNA. The comparison shows that the compaction rates can differ by a factor of up to 1000 for the same amount of histones, depending on the system used and on the presence of histone tails, which can be subjected to post-translational modifications. Reactions with purified reconstitution systems follow a slow and sequential mechanism, compatible with the deposition of one (H3-H4)(2) tetramer followed by two (H2A-H2B) dimers. Addition of the histone chaperone NAP-1 increases both the rate of the reaction and the packing ratio of the final product. These stimulatory effects cannot be obtained with polyglutamic acid or RNA, suggesting that yNAP-1 impact on the reaction cannot simply be explained in terms of charge screening. Faster compaction kinetics and higher packing ratios are reproducibly reached with extracts, indicating a role of additional components present in this system. Data are discussed and models proposed to account for the kinetics obtained in our single-molecule assay.

FIGURE 1

Schematic of the experimental apparatus. Syringe pump (1), electro-valve (2), inlet channel (I), outlet channel 1 (O1), and outlet channel 2 (O2). (A) Biological samples are aspirated in microchannel O1 until the concentration is homogeneous at the T-junction. (B) The electrovalve switches the aspiration to channel O2 where the objective is positioned. (C) Example of compaction of an individual DNA molecule in time in the presence of 2 ng/μL of native histones.

FIGURE 2

Chromatin assembly kinetics with Xenopus egg extracts appears as a one-step process. Xenopus egg extracts were diluted to 1:50, 1:100, 1:150, and 1:300 (from left to right on the graph) in a buffer containing 1 mM MgCl₂ and 0.5 mM ATP. Each curve is fitted with a single exponential, and only one fitting parameter is adjusted for all the curves.

FIGURE 3

Nucleosome reconstitution with native, WT, and tailless recombinant histones with yNAP-1 follows a three-step kinetics. (A) Nucleosome reconstitution with WT recombinant histones for three different concentrations (, , and 1.4 ng/μL) with a yNAP-1/octamer molar ratio of 0.6:1. The curves are fitted with a three-step kinetics model (solid lines). (B) Fitting of the reconstitution curve (3 ng/μL of WT recombinant histones) with a one-step kinetic model (dashed line) or a three-step kinetic model (solid line). (C) Comparison of the reconstitution kinetics with WT recombinant (1), native (2) and tailless recombinant (3) histones. The histone concentration is set at 2 ng/μL and the yNAP-1/octamer molar ratio is 0.6:1.

FIGURE 4

Role of yNAP-1 in the kinetics of nucleosome reconstitution. (A) Assembly kinetics with native histones (2 ng/μL) in the presence (diamonds) or absence (dotted line) of yNAP-1. (B) Assembly kinetics with WT recombinant histones (2 ng/μL) in the absence (orange) or presence of yNAP-1 at a molar ratio of 0.6:1 (blue) or 6:1 (dash-dotted line). Assembly kinetics with tailless recombinant histones with yNAP-1 at a molar ratio of 0.6:1 (green) or without yNAP-1 (red).

FIGURE 5

PGA and RNA do not facilitate nucleosome reconstitution. Comparison of the reconstitution with extracts (1) or native histones (2ng/μL) + [yNAP-1 – 0.6:1 molar ratio (2), no chaperone (3), RNA – 2:1 mass ratio (4), PGA – 5:1 mass ratio (5)].

FIGURE 6

Packing ratio is >25 with extracts and ∼6–7 with purified proteins. The assembly with extracts (1) at 2 ng/μL yields a compact structure (folding ratio >25). The length of the chromatin fiber assembled with yNAP-1 + WT recombinant (2), yNAP-1 + native (3), and yNAP-1 + tailless recombinant (4) histones, at a concentration of 2 ng/μL, reaches a plateau (seen only for WT recombinant histones because of the time axis truncation) from which we deduce the folding ratio. For native histones, it takes 30 s to reach the final length and for tailless histones 50 s.

FIGURE 7

(A) Raw compaction curves obtained with extracts at two different positions along the channel (solid circles, x = 2 mm; shaded crosses, x = 3 mm). The two curves appear different because the extracts take more time to arrive in the vicinity of the molecule located further in the channel. The plots of the corresponding casein concentration profiles are given as dotted lines (x = 2 mm, solid; x = 3 mm, shaded). (B) The same compaction curves as in A after data treatment: the normalized length is plotted versus the pertinent time τ (for the solid curve, τ(2 mm,t) ; for the shaded curve, τ(3 mm,t)).