Immunological characterization of chromatin assembly factor I, a human cell factor required for chromatin assembly during DNA replication in vitro

Chromatin assembly factor I (CAF-I) is a multisubunit protein complex purified from the nuclei of human cells and required for chromatin assembly during DNA replication in vitro. Purified CAF-I promotes chromatin assembly in a reaction that is dependent upon, and coupled with, DNA replication and is therefore likely to reflect events that occur during S phase in vivo. In order to investigate the regulation and mechanism of CAF-I and the replication-dependent chromatin assembly process, we have used the purified protein to raise monoclonal antibodies. In this report we describe the characterization of a panel of monoclonal antibodies which recognize different subunits of the CAF-I complex. We use immunoprecipitation analysis to show that CAF-I exists as a multiprotein complex in vivo and that some of the polypeptides are phosphorylated. In addition, immunocytochemistry demonstrates that CAF-I is localized to the nucleus of human cells. Finally, monoclonal antibodies directed against the individual subunits of CAF-I immunodeplete chromatin assembly activity from nuclear extracts, confirming that CAF-I is a multisubunit protein required for chromatin assembly in vitro.     

Stepwise assembly of chromatin during DNA replication in vitro.

A cell free system that supports replication-dependent chromatin assembly has been used to determine the mechanism of histone deposition during DNA replication. CAF-I, a human cell nuclear factor, promotes chromatin assembly on replicating SV40 DNA in the presence of a crude cytosol replication extract. Biochemical fractionation of the cytosol extract has allowed separation of the chromatin assembly reaction into two steps. During the first step, CAF-I targets the deposition of newly synthesized histones H3 and H4 to the replicating DNA. This reaction is dependent upon and coupled with DNA replication, and utilizes the newly synthesized forms of histones H3 and H4, which unlike bulk histone found in chromatin, do not bind to DNA by themselves. The H3/H4-replicated DNA complex is a stable intermediate which exhibits a micrococcal nuclease resistant structure and can be isolated by sucrose gradient sedimentation. In the second step, this replicated precursor is converted to mature chromatin by the addition of histones H2A and H2B in a reaction that can occur after DNA replication. The requirement for CAF-I in at least the first step of the reaction suggests a level of cellular control for this fundamental process.     

Asf1 is required for viability and chromatin assembly during DNA replication in vertebrate cells

Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1- and HIRA-independent chromatin-modulating process for cell proliferation.     

Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro.

Cell extracts (S100) derived from human 293 cells were separated into five fractions by phosphocellulose chromatography and monitored for their ability to support simian virus 40 (SV40) DNA replication in vitro in the presence of purified SV40 T antigen. Three fractions, designated I, IIA, and IIC, were essential. Fraction IIC contained the known replication factors topoisomerases I and II, but in addition contained a novel replication factor called RF-C. The RF-C activity, assayed in the presence of I, IIA, and excess amounts of purified topoisomerases, was detected in both cytosol and nuclear fractions, but was more abundant in the latter fraction. RF-C was purified from the 293 cell nuclear fraction to near homogeneity by conventional column chromatography. The reconstituted reaction mix containing purified RF-C could replicate SV40 origin-containing plasmid DNA more efficiently than could the S100 extract, and the products were predominantly completely replicated, monomer molecules. Interestingly, in the absence of RF-C, early replicative intermediates accumulated and subsequent elongation was aberrant. Hybridization studies with strand-specific, single-stranded M13-SV40 DNAs showed that in the absence of RF-C, abnormal DNA synthesis occurred preferentially on the lagging strand, and leading-strand replication was inefficient. These products closely resembled those previously observed for SV40 DNA replication in vitro in the absence of proliferating-cell nuclear antigen. These results suggest that an elongation complex containing RF-C and proliferating-cell nuclear antigen is assembled after formation of the first nascent strands at the replication origin. Subsequent synthesis of leading and lagging strands at a eucaryotic DNA replication fork can be distinguished by different requirements for multiple replication components, but we suggest that even though the two polymerases function asymmetrically, they normally progress coordinately.     

Purification and properties of a host cell protein required for poliovirus replication in vitro

A host cell protein required for poliovirus RNA-dependent RNA replicase activity in vitro has been purified several thousand-fold from an uninfected HeLa cell postmitochondrial supernatant. A single protein of apparent Mr = approximately 67,000 daltons and pI 6.3 is associated with this "host factor" activity. Poly(U)-Sepharose chromatography of the template-dependent replicase isolated from poliovirus-infected cells results in the complete loss of replicase activity if a salt gradient is used to develop the column. Host factor elutes early in the salt gradient and restores replicase activity to protein fractions eluted later in the gradient. The host factor, estimated to be present at 50,000-100,000 copies/cell, interacts physically with replicase.     

Chromatin assembly during SV40 DNA replication in vitro

A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen. Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA. The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract. The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I. Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure. These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes.     

Purification of ribonuclease H as a factor required for initiation of in vitro Co1E1 DNA replication.

Escherichia coli ribonuclease H was purified to near-homogeneity and identified as the only additional factor required for initiation of in vitro Co1E1 DNA replication from the unique origin by RNA polymerase and DNA polymerase I. Both ribonuclease H activity and stimulating activity for Co1E1 DNA synthesis comigrate with the single protein band in gel electrophoresis. These two activities coincide throughout the process of purification. Some DNA synthesis takes place on covalently closed-circular DNA molecules other than Co1E1 DNA with the three purified enzymes. This DNA synthesis is suppressed by an Escherichia coli single-strand DNA binding protein and/or a high concentration of ribonuclease H. Negative superhelicity of template DNA is required for efficient primer formation. No evidence that supports involvement of ribonuclease III in initiation of Co1E1 DNA replication or its regulation was found.     

Studies on DNA Topoisomerase activity during in vitro chromatin assembly

The in vitro assembly of chromatin, promoted by the Xenopus cell-free extract (S-150), can be inhibited by oxolinic acid and to a lesser extent by nalidixic acid. Both of these antibiotics have been shown to block the activity of the specialized type 11 Topoisomerase, bacterial DNA Gyrase. Oxolinic acid induces a DNA cleavage by Micrococcal Nuclease at specific sequences in the multiple cloning vector pGEM-4. Nalidixic acid does not inhibit DNA supercoiling, but does diminish the extent of chromatin formation achieved by the S-150 on circular DNA templates. The Topoisomerase I inhibitor, berenil, does not inhibit extensive chromatin assembly, although it aloes diminish the level of supercoiling. Taken together, these results suggest that both topoisomerases play a role in the assembly process. Topoisomerase I may catalyze both the introduction of unconstrained supercoils into relaxed DNA and the formation of monosomes, while Topoisomerase 11 may promote extended chromatin assembly.     

Contribution of CAF-I to anaphase-promoting-complex-mediated mitotic chromatin assembly in Saccharomyces cerevisiae

The anaphase-promoting complex (APC) is required for mitotic progression and genomic stability. Recently, we demonstrated that the APC is also required for mitotic chromatin assembly and longevity. Here, we investigated the role the APC plays in chromatin assembly. We show that apc5(CA) mutations genetically interact with the CAF-I genes as well as ASF1, HIR1, and HIR2. When present in multiple copies, the individual CAF-I genes, CAC1, CAC2, and MSI1, suppress apc5(CA) phenotypes in a CAF-1- and Asf1p-independent manner. CAF-I and the APC functionally overlap, as cac1delta cac2delta msi1delta (caf1delta) cells expressing apc5(CA) exhibit a phenotype more severe than that of apc5(CA) or caf1delta. The Ts- phenotypes observed in apc5(CA) and apc5(CA) caf mutants may be rooted in compromised histone metabolism, as coexpression of histones H3 and H4 suppressed the Ts- defects. Synthetic genetic interactions were also observed in apc5(CA) asf1delta cells. Furthermore, increased expression of genes encoding Asf1p, Hir1p, and Hir2p suppressed the apc5(CA) Ts- defect in a CAF-I-dependent manner. Together, these results suggest the existence of a complex molecular mechanism controlling APC-dependent chromatin assembly. Our data suggest the APC functions with the individual CAF-I subunits, Asf1p, and the Hir1p and Hir2p proteins. However, Asf1p and an intact CAF-I complex are dispensable for CAF-I subunit suppression, whereas CAF-I is necessary for ASF1, HIR1, and HIR2 suppression of apc5(CA) phenotypes. We discuss the implications of our observations.     

CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA

Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric alpha-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.     

Essential role of chromatin assembly factor-1-mediated rapid nucleosome assembly for DNA replication and cell division in vertebrate cells

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-gamma are required for cell viability. These observations highlighted the essential role of CAF-1-dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.     

cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication.

RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.     

Postreplicative chromatin assembly by Drosophila and human chromatin assembly factor 1.

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.     

Requirement of Cyclin/Cdk2 and protein phosphatase 1 activity for chromatin assembly factor 1-dependent chromatin assembly during DNA synthesis

The influence of reversible protein phosphorylation on nucleosome assembly during DNA replication was analyzed in extracts from human cells. Inhibitor studies and add-back experiments indicated requirements of cyclin A/Cdk2, cyclin E/Cdk2, and protein phosphatase type 1 (PP1) activities for nucleosome assembly during DNA synthesis by chromatin assembly factor 1 (CAF-1). The p60 subunit of CAF-1 is a molecular target for reversible phosphorylation by cyclin/Cdk complexes and PP1 during nucleosome assembly and DNA synthesis in vitro. Purified p60 can be directly phosphorylated by purified cyclin A/Cdk2, cyclin E/Cdk2, and cyclin B1/Cdk1, but not by cyclin D/Cdk4 complexes in vitro. Cyclin B1/Cdk1 triggers hyperphosphorylation of p60 in the presence of additional cytosolic factors. CAF-1 containing hyperphosphorylated p60 prepared from mitotic cells is inactive in nucleosome assembly and becomes activated by dephosphorylation in vitro. These data provide functional evidence for a requirement of the cell cycle machinery for nucleosome assembly by CAF-1 during DNA replication.     

Chromatin assembly. Relationship of chromatin structure to DNA sequence during simian virus 40 replication

The accessibility of five specific DNA sequences to six different single site restriction endonucleases was evaluated in replicating and mature simian virus 40 chromosomes isolated by three different methods. Electron microscopic and gel electrophoretic analysis of the DNA digestion products demonstrated that DNA accessibility in chromatin was established within 400 base pairs of replication forks and remained essentially unchanged during production of mature chromosomes and their subsequent re-entry into the replication pool. Saturating amounts of each enzyme reproducibly cut a fraction of the chromosomes, ranging from 13 to 49%. This is consistent with a nearly random phasing of chromatin structure. Examples in which all chromosomes were either cleaved or intact were never observed. Although variation in the accessibility of DNA sites near the origin of replication could be interpreted as preferred phasing in about 25% of the chromosomes, the finding that two isoschizomers, Hpa II and Msp I, did not cut chromosomes to the same extent precludes an unambiguous interpretation of the extents of cleavage of individual restriction enzymes. Since the extent of DNA cleavage observed at each restriction site was essentially indistinguishable in replicating as compared to mature chromosomes, the accessibility of DNA sequences near the origin is not obviously related to replication. Furthermore, the accessibility of DNA sites on one arm of a single replication fork was the same as the homologous sites on the other arm, consistent with a nearly random phasing of chromatin structure on both arms. This suggests that chromatin assembly occurs independently on the 2 sibling molecules of a single replicating chromosome.