Multistep Chromatin Assembly on Supercoiled Plasmid DNA by Nucleosome Assembly Protein-1 and ATP-utilizing Chromatin Assembly and Remodeling Factor

Abstract

We examine in vitro nucleosome assembly by nucleosome assembly protein-1 (NAP-1) and ATP-utilizing chromatin assembly and remodeling factor (ACF). In contrast to previous studies that used relaxed, circular plasmids as templates, we have found that negatively supercoiled templates reveal the distinct roles of NAP-1 and ACF in histone deposition and the formation of an ordered nucleosomal array. NAP-1 can efficiently deposit histones onto supercoiled plasmids. Furthermore, NAP-1 exhibits a greater affinity for histones H2A-H2B than does naked DNA, but in the presence of H3-H4, H2A-H2B are transferred from NAP-1 to the plasmid templates. These observations underscore the importance of a high affinity between H2A-H2B and NAP-1 for ordered transfer of core histones onto DNA. In addition, recombinant ACF composed of imitation switch and Acf1 can extend closely packed nucleosomes, which suggests that recombinant ACF can mobilize nucleosomes. In the assembly reaction with a supercoiled template, ACF need not be added simultaneously with NAP-1. Regularly spaced nucleosomes are generated even when recombinant ACF is added after core histones are transferred completely onto the DNA. Atomic force microscopy, however, suggests that NAP-1 alone fails to accomplish the formation of fine nucleosomal core particles, which are only formed in the presence of ACF. These results suggest a model for the ordered deposition of histones and the arrangement of nucleosomes during chromatin assembly in vivo.The assembly of genomic DNA and histones into chromatin is a fundamental process of eukaryotes that affects a broad range of biological phenomena, including DNA replication, DNA repair, gene expression, and progression through the cell cycle (for reviews, see Refs. 1-7). The basic structural unit of chromatin is the nucleosome, consisting of 145 base pairs (bp) 1 of DNA wrapped around an octamer of the core histones H2A, H2B, H3, and H4. Crystallographic studies have shown that an H3-H4 tetramer occupies the central region of the nucleosome, and the H2A-H2B dimers bind to the peripheral region (8, 9). Thus, it is implicit in the structure of the nucleosome that H3 and H4 must be deposited prior to the incorporation of H2A and H2B. In agreement with this, several studies of histone transfer and of chromatin assembly during DNA replication have indicated that H3-H4 deposition occurs first, then H2A-H2B, followed by linker histones (such as histone H1) and the establishment of the ordered nucleosomal arrays that are characteristic of bulk chromatin in vivo (for reviews, see Refs. 10 -13).Biochemical studies have led to the identification of proteins that mediate the reconstitution of core histones into nucleosomes. Nearly all of these factors are core histone-binding proteins that contain stretches of acidic amino acid residues. Some of these histone chaperones, such as nucleoplasmin and nucleosome assembly protein-1 (NAP-1), exhibit a preference for binding to histones H2A and H2B relative to histones H3 and H4. Other histone chaperones,...

Dynamics of Nucleosome Assembly and Effects of DNA Methylation

“…We would have observed FRET signals with a wide range of efficiencies if dimers randomly but stably bound to DNA, which would result in DNA random coiling. This result strongly suggests that a tetramer must bind DNA before dimers can be stably incorporated, which is in good agreement with previous studies (14,39). Fig.…”

Structural evidence for Nap1‐dependent H2A–H2B deposition and nucleosome assembly

“…The DNA binding surfaces are conserved in other H2A variants such as H2A.Z, and therefore, our structure also explains why Nap1 can assist remodeling complexes such as SWR1 in incorporating H2A.Z into chromatin (Kobor et al , 2004; Mizuguchi et al , 2004; Park et al , 2005; Luk et al , 2007). This DNA binding region is conserved in H3–H4 as well, which likely explains why yNap1 can interact with all four core histones in vitro (McQuibban et al , 1998; Mosammaparast et al , 2001; Nakagawa et al , 2001; Bowman et al , 2011). However, we find no evidence that yNap1 chaperones H3–H4 in vivo .…”

Assembly of single chromatin fibers depends on the tension in the DNA molecule: Magnetic tweezers study

“…NAP-1 is known to assemble uniformly spaced nucleosomes of a short repeat length (26,28,29). Although additional ATP-dependent factors are reportedly needed to achieve physiologically relevant nucleosome spacing (30), the actual formation of nucleosomes requires only NAP-1. Because it is the initial nucleosome assembly on naked DNA that we are interested in (rather than the maturation of the fiber structure), we performed our experiments with NAP-1 only.…”