Postreplicative Chromatin Assembly by Drosophila and Human Chromatin Assembly Factor 1

Abstract

To study the relationship between DNA replication and chromatin assembly, we have purified a factor termed Drosophila chromatin assembly factor 1 (dCAF-1) to approximately 50% homogeneity from a nuclear extract derived from embryos. dCAF-1 appears to consist of four polypeptides with molecular masses of 180, 105, 75, and 55 kDa. dCAF-1 preferentially mediates chromatin assembly of newly replicated DNA relative to unreplicated DNA during T-antigen-dependent simian virus 40 DNA replication in vitro, as seen with human CAF-1. Analysis of the mechanism of DNA replication-coupled chromatin assembly revealed that both dCAF-1 and human CAF-1 mediate chromatin assembly preferentially with previously yet newly replicated DNA relative to unreplicated DNA. Moreover, the preferential assembly of the postreplicative DNA was observed at 30 min after inhibition of DNA replication by aphidicolin, but this effect slowly diminished until it was no longer apparent at 120 min after inhibition of replication. These findings suggest that the coupling between DNA replication and chromatin assembly may not necessarily involve a direct interaction between the replication and assembly factors at a replication fork.Replication of the eukaryotic genome involves both the replication of DNA and the assembly of the newly synthesized DNA into chromatin (for reviews, see references 7, 11, 34, 35, and 36), and it is therefore important to study the relationship between DNA replication and chromatin assembly. The current data suggest that the assembly of chromatin in vivo begins immediately after DNA replication by a mechanism that leads to a random distribution of the newly synthesized and preexisting histones between the daughter DNA strands. In addition, it appears that nucleosome assembly occurs by the initial deposition of histones H3 and H4 followed by the incorporation of histones H2A and H2B. Structures that resemble bulk nucleosomes have been observed at replication forks by elec-

The enhancer of trithorax and polycomb gene Caf1/p55 is essential for cell survival and patterning in Drosophila development

“…Although our results suggest that disruption of PRC2 function may be the most important consequence of Caf1 gain-or loss-of-function, many phenotypes we observed in Caf1 mutant tissue are also reminiscent of mutations in members of other complexes previously shown to contain Caf1. It is not surprising that all three alleles of Caf1 in the current study are homozygous lethal, and that Caf1 short cells have poor viability, considering that Caf1 has been found in the NURF and CAF-1 complexes, which have fundamental roles in nucleosome assembly and spacing (Bulger et al, 1995;Kamakaka et al, 1996;Martinez-Balbas et al, 1998;Tyler et al, 1996;Verreault et al, 1996). The apoptosis we observe in eyes with Caf1 short clones is also consistent with a role for Caf1 in the dREAM (Drosophila Rbf, E2F2, and Myb-interacting proteins) complex.…”

The Drosophila DAXX-Like Protein (DLP) Cooperates with ASF1 for H3.3 Deposition and Heterochromatin Formation

“…Numerous proteins were found to be associated with e-H3 and e-H3.3. Mass spectrometry and immunoblot analyses led to the identification of several common components of the e-H3 and e-H3.3 complexes, including the core histones H2A, H2B, H3, and H4 and the well-characterized histone chaperones ASF1 (27), NAP1 (nucleosome assembly protein 1) (28), Drosophila NASP (nuclear autoantigenic sperm protein) homolog (29), and Drosophila CAF-1 (chromatin assembly factor 1) (p105/p180 subunits) (30) (Fig. 2A, left).…”

Chromatin remodelling and DNA replication: from nucleosomes to loop domains

“…Deposition of nucleosomes onto newly replicated DNA is mediated by histone chaperones, such as NAP-1 (reviewed in Ito et al, 1997) andCAF-1 (Ridgway andAlmouzni, 2000), which interact with histones and stimulate their association with newly replicated DNA. CAF-1 is composed of three highly conserved sub-units (Bulger et al, 1995;Kamakaka et al, 1996). The largest subunit of CAF-1, p150, has been shown to interact with the Proliferating Cell Nuclear Antigen (PCNA) (Shibahara and Stillman, 1999).…”