The p55 subunit of Drosophila chromatin assembly factor 1 is homologous to a histone deacetylase-associated protein.

To gain a better understanding of DNA replication-coupled chromatin assembly, we have isolated the cDNA encoding the smallest (apparent molecular mass, 55 kDa; termed p55) subunit of Drosophila melanogaster chromatin assembly factor 1 (dCAF-1), a multisubunit protein that is required for the assembly of nucleosomes onto newly replicated DNA in vitro. The p55 polypeptide comprises seven WD repeat motifs and is homologous to the mammalian RbAp48 protein, which is associated with the HD1 histone deacetylase. dCAF-1 was immunopurified by using affinity-purified antibodies against p55; the resulting dCAF-1 preparation possessed the four putative subunits of dCAF-1 (p180, p105, p75, and p55) and was active for DNA replication-coupled chromatin assembly. Moreover, dCAF-1 activity was specifically depleted with antibodies against p55. Thus, p55 is an integral component of dCAF-1. p55 is localized to the nucleus and is present throughout Drosophila development. Consistent with the homology between p55 and the HD1-associated RbAp48 protein, histone deacetylase activity was observed to coimmunoprecipitate specifically with p55 from a Drosophila nuclear extract. Furthermore, a fraction of the p55 protein becomes associated with the newly assembled chromatin following DNA replication. These findings collectively suggest that p55 may function as a link between DNA replication-coupled chromatin assembly and histone modification.     

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.     

CAF-1 is essential for Drosophila development and involved in the maintenance of epigenetic memory

DNA synthesis during S-phase and upon DNA repair is accompanied by chromatin assembly. The chromatin assembly factor CAF-1 has been biochemically well-characterized to deposit histones onto newly synthesized DNA. To gain insights into the in vivo functions of CAF-1 in Drosophila, we generated null mutants of the largest subunit of dCAF-1, dCAF-1-p180. We show that, unlike CAF-1 mutant yeast, dCAF-1-p180 mutant flies are hemizygous lethal. Removal of maternal dCAF-1-p180 activity by germline clones blocks oogenesis. Tissue-specific deletion of dCAF-1-p180 in the eye primordia disrupts eye development. In addition, reduction of dCAF-1-p180 activity suppresses gene silencing at heterochromatin and antagonizes Polycomb-mediated cell fate determination. Furthermore, heterozygous dCAF-1-p180 mutant flies display an increased sensitivity to γ-irradiation and a reduced efficiency in recombinational double strand break (DSB) repair. Our experiments also show that human hCAF-1-p150 can rescue the dCAF-1-p180 mutant flies, demonstrating a functional conservation of eukaryotic CAF-1 activities in vivo. Together, our results establish that dCAF-1-p180 is an essential gene for Drosophila development and further underscore the importance of dCAF-1 in regulating gene expression and DNA repair in vivo.     

黑腹果蝇dCAF-1-p55突变引起果蝇发育迟缓和染色体不稳定性

在真核生物中高度保守的染色质装配因子1(chromatin assembly factor 1,CAF-1)是染色质装配过程中的组蛋白分子伴侣之一.dCAF-1-p55是果蝇中CAF-1复合物中的最小亚基,它与另外两个亚基dCAF-1-p180及dCAF-1-p105一起负责将组蛋白H3/H4组装到新合成的DNA上.除了CAF-1复合物,dCAF-1-p55还参与其他多个复合物的形成,如NURF、PRC2及Sin3-HDAC1.dCAF-1-p55的这一广泛参与性提示了其功能的多样性和重要性.为了研究dCAF-1-p55的体内功能,我们利用基因靶向敲除技术制备了果蝇dCAF-1-p55突变体.实验结果表明,dCAF-1-p55的缺失导致果蝇发育迟缓并且最终致死.进一步研究发现,在dCAF-1-p55突变细胞中,中期染色体较为松散,姐妹染色单体连接异常,后期染色体不能正常分离.这些缺陷都是与癌症发生密切相关的染色体不稳定性(chromosome instability,CIN)的典型特征.综上所述,我们的研究表明了dCAF-1-p55在果蝇发育过程及维持染色体稳定性方面的重要作用,同时提示该基因具有保护细胞免遭CIN和癌变的潜在功能.     

Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays.

We describe the cloning and analysis of Drosophila nucleosome assembly protein 1 (dNAP-1), a core histone-binding protein that functions with other chromatin assembly activities in a Drosophila chromatin assembly factor 1-containing fraction (dCAF-1 fraction) in the ATP-facilitated assembly of regularly spaced nucleosomal arrays from purified core histones and DNA. Purified, recombinant dNAP-1 acts cooperatively with a factor(s) in the dCAF-1 fraction in the efficient and DNA replication-independent assembly of chromatin. In the presence of histone H1, the repeat length of the chromatin is similar to that of native chromatin from Drosophila embryos. By coimmunoprecipitation analysis, dNAP-1 was found to be associated with histones H2A and H2B in a crude whole-embryo extract, which suggests that dNAP-1 is bound to the histones in vivo. Studies of the localization of dNAP-1 in the Drosophila embryo revealed that the factor is present in the nucleus during S phase and is predominantly cytoplasmic during G2 phase. These data suggest that NAP-1 acts as a core histone shuttle which delivers the histones from the cytoplasm to the chromatin assembly machinery in the nucleus. Thus, NAP-1 appears to be one component of a multifactor chromatin assembly machinery that mediates the ATP-facilitated assembly of regularly spaced nucleosomal arrays.     

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.     

Phosphorylation-Mediated Control of Histone Chaperone ASF1 Levels by Tousled-Like Kinases

Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs) and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK-mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases.     

Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway

The efficient assembly of newly replicated and repaired DNA into chromatin is essential for proper genome function. Based on genetic studies in Saccharomyces cerevisiae, the histone chaperone anti-silencing function 1 (Asf1) has been implicated in the DNA repair response. Here, the human homologs are shown to function synergistically with human CAF-1 to assemble nucleosomes during nucleotide excision repair in vitro. Furthermore, we demonstrate that hAsf1 proteins can interact directly with the p60 subunit of hCAF-1. In contrast to hCAF-1 p60, the nuclear hAsf1 proteins are not significantly associated with chromatin in cells before or after the induction of DNA damage, nor specifically recruited to damaged DNA during repair in a bead-linked DNA assay. A model is proposed in which the synergism between hAsf1 and CAF-1 for nucleosome formation during DNA repair is achieved through a transient physical interaction allowing histone delivery from Asf1 to CAF-1.     

Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities.

The yeast Saccharomyces cerevisiae catalytic DNA polymerase I 180-kDa subunit and the tightly associated 86-kDa polypeptide have been purified using immunoaffinity chromatography, permitting further characterization of the DNA polymerase activity of the DNA primase-DNA polymerase protein complex. The subunits were purified to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit. When the individual subunits were recombined in vitro a p86p180 physical complex formed spontaneously, as judged by immunoprecipitation of 180-kDa polypeptide and DNA polymerase activity with the anti-86-kDa monoclonal antibody. The 86-kDa subunit stabilized the DNA polymerase activity of the 180-kDa catalytic subunit at 30 degrees C, the physiological temperature. The apparent DNA polymerase processivity of 50-60 nucleotides on poly(dA).oligo(dT)12 or poly(dT).oligo(A)8-12 template-primer was not affected by the presence of the 86-kDa subunit but was reduced by increased Mg2+ concentration. The Km of the catalytic 180-kDa subunit for dATP or DNA primer terminus was unaffected by the presence of the 86-kDa subunit. The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis that had been observed previously in the DNA primase-DNA polymerase protein complex. The 180-kDa subunit possessed a 3'----5'-exonuclease activity that catalyzed degradation of polynucleotides, but degradation of oligonucleotide substrates of chain lengths up to 50 was not detected. This exonuclease activity was unaffected by the presence of the 86-kDa subunit. Despite the striking physical similarity of the DNA primase-DNA polymerase protein complex in all eukaryotes examined, the data presented here indicate differences in the enzymatic properties detected in preparations of the DNA polymerase subunits isolated from S. cerevisiae as compared with the properties of preparations from Drosophila cells. In particular, the 3'----5'-exonuclease activity associated with the yeast catalytic DNA polymerase subunit was not masked by the 86-kDa subunit.     

A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage

Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA-CAF-1 interaction in the context of DNA damage processing and checkpoint control.     

Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin

Chromatin assembly factor 1 (CAF-1) is required for inheritance of epigenetically determined chromosomal states in vivo and promotes assembly of chromatin during DNA replication in vitro. Herein, we demonstrate that after DNA replication, replicated, but not unreplicated, DNA is also competent for CAF-1-dependent chromatin assembly. The proliferating cell nuclear antigen (PCNA), a DNA polymerase clamp, is a component of the replication-dependent marking of DNA for chromatin assembly. The clamp loader, replication factor C (RFC), can reverse this mark by unloading PCNA from the replicated DNA. PCNA binds directly to p150, the largest subunit of CAF-1, and the two proteins colocalize at sites of DNA replication in cells. We suggest that PCNA and CAF-1 connect DNA replication to chromatin assembly and the inheritance of epigenetic chromosome states.     

Human CAF-1-dependent nucleosome assembly in a defined system

Replication-coupled nucleosome assembly is a critical step in packaging newly synthesized DNA into chromatin. Previous studies have defined the importance of the histone chaperones CAF-1 and ASF1A, the replicative clamp PCNA, and the clamp loader RFC for the assembly of nucleosomes during DNA replication. Despite significant progress in the field, replication-coupled nucleosome assembly is not well understood. One of the complications in elucidating the mechanisms of replication-coupled nucleosome assembly is the lack of a defined system that faithfully recapitulates this important biological process in vitro. We describe here a defined system that assembles nucleosomal arrays in a manner dependent on the presence of CAF-1, ASF1A-H3-H4, H2A-H2B, PCNA, RFC, NAP1L1, ATP, and strand breaks. The loss of CAF-1 p48 subunit causes a strong defect in packaging DNA into nucleosomes by this system. We also show that the defined system forms nucleosomes on nascent DNA synthesized by the replicative polymerase δ. Thus, the developed system reproduces several key features of replication-coupled nucleosome assembly.     

Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.     

Molecular Architecture of the Mouse DNA Polymerase α-Primase Complex

The DNA polymerase alpha-primase complex is the only enzyme that provides RNA-DNA primers for chromosomal DNA replication in eukaryotes. Mouse DNA polymerase alpha has been shown to consist of four subunits, p180, p68, p54, and p46. To characterize the domain structures and subunit requirements for the assembly of the complex, we constructed eukaryotic polycistronic cDNA expression plasmids expressing pairwise the four subunits of DNA polymerase alpha. In addition, the constructs contained an internal ribosome entry site derived from poliovirus. The constructs were transfected in different combinations with vectors expressing single subunits to allow the simultaneous expression of three or four of the subunits in cultured mammalian cells. We demonstrate that the carboxyl-terminal region of p180 (residues 1235 to 1465) is essential for its interaction with both p68 and p54-p46 by immunohistochemical analysis and coprecipitation studies with antibodies. Mutations in the putative zinc fingers present in the carboxyl terminus of p180 abolished the interaction with p68 completely, although the mutants were still capable of interacting with p54-p46. Furthermore, the amino-terminal region (residues 1 to 329) and the carboxyl-terminal region (residues 1280 to 1465) were revealed to be dispensable for DNA polymerase activity. Thus, we can divide the p180 subunit into three domains. The first is the amino-terminal domain (residues 1 to 329), which is dispensable for both polymerase activity and subunit assembly. The second is the minimal core domain (residues 330 to 1279), required for polymerase activity. The third is the carboxyl-terminal domain (residues 1280 to 1465), which is dispensable for polymerase activity but required for the interaction with the other three subunits. Taken together, these results allow us to propose the first structural model for the DNA polymerase alpha-primase complex in terms of subunit assembly, domain structure, and stepwise formation at the cellular level.     

The Second-Largest Subunit of the Mouse DNA Polymerase α-Primase Complex Facilitates Both Production and Nuclear Translocation of the Catalytic Subunit of DNA Polymerase α

DNA polymerase α-primase is a replication enzyme necessary for DNA replication in all eukaryotes examined so far. Mouse DNA polymerase α is made up of four subunits, the largest of which is the catalytic subunit with a molecular mass of 180 kDa (p180). This subunit exists as a tight complex with the second-largest subunit (p68), whose physiological role has remained unclear up until now. We set out to characterize these subunits individually or in combination by using a cDNA expression system in cultured mammalian cells. Coexpression of p68 markedly increased the protein level of p180, with the result that ectopically generated DNA polymerase activity was dramatically increased. Immunofluorescence analysis showed that while either singly expressed p180 or p68 was localized in the cytoplasm, cotransfection of both subunits resulted in colocalization in the nucleus. We identified a putative nuclear localization signal for p180 (residues 1419 to 1437) and found that interaction with p68 is essential for p180 to translocate into the nucleus. These results indicate that association of p180 with p68 is important for both protein synthesis of p180 and translocation into the nucleus, implying that p68 plays a pivotal role in the newly synthesized DNA polymerase α complex.