Abstract
To gain a better understanding of the factors that can mediate chromatin assembly, we have purified and cloned a core histone-binding protein from Drosophila melanogaster embryos. This protein resembles Xenopus laevis nucleoplasmin, and it has therefore been termed dNLP, for Drosophila nucleoplasmin-like protein. dNLP is a nuclear protein that is present throughout development. Both purified native and recombinant dNLP bind to core histones and can function in the assembly of approximately regularly spaced nucleosomal arrays in a reaction that additionally requires DNA, purified core histones, ATP, and a partially purified fraction (containing at least one other assembly activity). We also analyzed the properties of an N-terminally truncated version of dNLP, termed dNLP-S, and found that the deletion of the N-terminal 31 residues of dNLP results in a loss of the specificity of the interaction of dNLP with core histones. We then compared the abilities of dNLP and Drosophila nucleosome assembly protein-1 (dNAP-1) to promote the decondensation of Xenopus sperm chromatin, a process that can be mediated by nucleoplasmin. We observed that dNAP-1, but not dNLP, was able to promote the decondensation of sperm chromatin. These and other data collectively suggest that dNLP may participate in parallel with other histone-binding proteins such as dNAP-1 in the assembly of chromatin.
Highlights
Chromatin assembly is a fundamental process that is involved in a broad range of biological phenomena such as gene regulation, recombination, DNA repair, and progression through the cell cycle, and it is important to investigate the factors that participate in the formation of chromatin
The ATP-utilizing chromatin assembly factor(s) in the dCAF-1 fraction has not yet been identified, it is known that the Drosophila homologue of chromatin assembly factor-1 (CAF-1) is
Purification and Cloning of Drosophila nucleoplasmin-like protein (dNLP)—Further analysis of the dCAF-4 fraction [29], from which we had purified and cloned Drosophila homologue of NAP-1 (dNAP-1) [21], led to the identification of a second, less abundant protein with an apparent molecular mass of 22 kDa that appeared to bind to core histones
Summary
Chromatin assembly is a fundamental process that is involved in a broad range of biological phenomena such as gene regulation, recombination, DNA repair, and progression through the cell cycle, and it is important to investigate the factors that participate in the formation of chromatin (for reviews, see Refs. 1–7). The current data suggest that nucleoplasmin and NAP-1 interact preferentially with H2A and H2B relative to H3 and H4 [17, 19, 21] and that N1/N2 and CAF-1 bind to H3 and H4 [14, 15, 17, 23, 24] These factors may act, at least in part, as histone chaperones that deliver the core histones to the newly replicated DNA. The mechanism of chromatin assembly is likely, to be more complex than the random deposition of histones that is mediated by the core histone-binding factors alone It is known from studies of chromatin assembly activities in crude extracts derived from Xenopus laevis oocytes [25], HeLa cells [26], or Drosophila melanogaster embryos [27, 28] that the assembly of approximately regularly spaced nucleosomal arrays is an ATP-dependent process. The ATP-utilizing chromatin assembly factor(s) in the dCAF-1 fraction has not yet been identified, it is known that the Drosophila homologue of CAF-1 (termed dCAF-1 protein, which should not be confused with the dCAF-1 fraction) is
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