P150 Subunit Research Articles

Comparative proteomics analysis of global cellular stress responses to hydroxyurea-induced DNA damage in HeLa cells.

Both environmental agents and spontaneous cellular events cause serious DNA damage, threatening the integrity of the genome. In response to replication stress or genotoxic agents triggered DNA damage, degradation of p12 subunit of DNA polymerase delta (Pol δ) results in an inter-conversion between heterotetramer (Pol δ4) and heterotrimer (Pol δ3) forms and plays a significant role in DNA damage response in eukaryotic cells. In this work, we used mass spectrometry-based proteomic approach to identify those cellular stress response protein changes corresponding to the degradation of p12 in DNA-damaged HeLa cells by the treatment with hydroxyurea (HU). A total of 736±13 proteins in non-treated control group and 741±19 protein spots in HU-treated cells were detected, of which 34 proteins (17 up-regulated and 17 down-regulated) exhibited significantly altered protein expression levels. Their physiological roles are mainly associated with cellular components, molecular functions, and biological processes by gene ontology analysis, among which 21 proteins were mapped to KEGG pathways. They are involved in 5 primary pathways with the subsets involving 16 secondary pathways by further KEGG analysis. More interestingly, the up-regulation of translationally controlled tumor protein was further identified to be associated with p12 degradation by Western blot analysis. Our works may enlarge and broaden our view for deeply understanding how global cellular stress responds to DNA damage, which could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.

Katanin p80 Regulates Human Cortical Development by Limiting Centriole and Cilia Number

Katanin is a microtubule-severing complex whose catalytic activities are well characterized, but whose invivo functions are incompletely understood. Human mutations in KATNB1, which encodes the noncatalytic regulatory p80 subunit of katanin, cause severe microlissencephaly. Loss of Katnb1 in mice confirms essential roles in neurogenesis and cell survival, while loss of zebrafish katnb1 reveals specific roles for katnin p80 in early and late developmental stages. Surprisingly, Katnb1 null mutant mouse embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. KATNB1-deficient human cells show defective proliferation and spindle structure, while Katnb1 null fibroblasts also demonstrate a remarkable excess of centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal unexpected functions for KATNB1 inregulating overall centriole, mother centriole, and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.

Expression of the p12 subunit of human DNA polymerase δ (Pol δ), CDK inhibitor p21WAF1, Cdt1, cyclin A, PCNA and Ki-67 in relation to DNA replication in individual cells

We recently reported that the p12 subunit of human DNA polymerase δ (Pol δ4) is degraded by CRL4Cdt2 which regulates the licensing factor Cdt1 and p21WAF1 during the G1 to S transition. Presently, we performed multiparameter laser scanning cytometric analyses of changes in levels of p12, Cdt1 and p21WAF1, detected immunocytochemically in individual cells, vis-à-vis the initiation and completion of DNA replication. The latter was assessed by pulse-labeling A549 cells with the DNA precursor ethynyl-2′-deoxyribose (EdU). The loss of p12 preceded the initiation of DNA replication and essentially all cells incorporating EdU were p12 negative. Completion of DNA replication and transition to G2 phase coincided with the re-appearance and rapid rise of p12 levels. Similar to p12 a decline of p21WAF1 and Cdt1 was seen at the end of G1 phase and all DNA replicating cells were p21WAF1 and Cdt1 negative. The loss of p21WAF1 preceded that of Cdt1 and p12 and the disappearance of the latter coincided with the onset of DNA replication. Loss of p12 leads to conversion of Pol δ4 to its trimeric form, Pol δ3, so that the results provide strong support to the notion that Pol δ3 is engaged in DNA replication during unperturbed progression through the S phase of cell cycle. Also assessed was a correlation between EdU incorporation, likely reflecting the rate of DNA replication in individual cells, and the level of expression of positive biomarkers of replication cyclin A, PCNA and Ki-67 in these cells. Of interest was the observation of stronger correlation between EdU incorporation and expression of PCNA (r = 0.73) than expression of cyclin A (r = 0.47) or Ki-67 (r = 0.47).

Extended String Binding Mode of the Phosphorylated Transactivation Domain of Tumor Suppressor p53

The transactivation domain (TAD) of tumor suppressor p53 has homologous subdomains, TAD1 and TAD2. Both are intrinsically disordered in their free states, but all structures of TAD1 and TAD2 bound to their target proteins have demonstrated use of an amphipathic α-helix, suggesting that the binding-coupled helix folding mechanism of TAD1 and TAD2 is essential. Although phosphorylation of TAD is important to switch the function of p53, bound structures of phosphorylated TAD1 and TAD2 have not been determined. Here, we reveal the recognition mechanism of the phosphorylated TAD2 bound to a pleckstrin homology (PH) domain from human TFIIH subunit p62 in an extended string-like conformation. This string-like binding mode of TAD2 seems to be independent of its phosphorylation in spite of enhanced binding activity upon phosphorylation. This is in contrast to the amphipathic helical binding mode of the unphosphorylated TAD2 to the yeast tfb1 PH domain and demonstrates that the p53 TAD2 has much higher conformational malleability than previously appreciated.

Dynactin functions as both a dynamic tether and brake during dynein-driven motility.

Dynactin is an essential cofactor for most cellular functions of the microtubule motor cytoplasmic dynein, but the mechanism by which dynactin activates dynein remains unclear. Here we use single molecule approaches to investigate dynein regulation by the dynactin subunit p150(Glued). We investigate the formation and motility of a dynein-p150(Glued) co-complex using dual-colour total internal reflection fluorescence microscopy. p150(Glued) recruits and tethers dynein to the microtubule in a concentration-dependent manner. Single molecule imaging of motility in cell extracts demonstrates that the CAP-Gly domain of p150(Glued) decreases the detachment rate of the dynein-dynactin complex from the microtubule and also acts as a brake to slow the dynein motor. Consistent with this important role, two neurodegenerative disease-causing mutations in the CAP-Gly domain abrogate these functions in our assays. Together, these observations support a model in which dynactin enhances the initial recruitment of dynein onto microtubules and promotes the sustained engagement of dynein with its cytoskeletal track.

A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli.

Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element-containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.

Mutant huntingtin affects cortical progenitor cell division and development of the mouse neocortex.

A polyglutamine expansion in huntingtin (HTT) causes the specific death of adult neurons in Huntington's disease (HD). Most studies have thus focused on mutant HTT (mHTT) toxicity in adulthood, and its developmental effects have been largely overlooked. We found that mHTT caused mitotic spindle misorientation in cultured cells by altering the localization of dynein, NuMA, and the p150(Glued) subunit of dynactin to the spindle pole and cell cortex and of CLIP170 and p150(Glued) to microtubule plus-ends. mHTT also affected spindle orientation in dividing mouse cortical progenitors, altering the thickness of the developing cortex. The serine/threonine kinase Akt, which regulates HTT function, rescued the spindle misorientation caused by the mHTT, by serine 421 (S421) phosphorylation, in cultured cells and in mice. Thus, cortical development is affected in HD, and this early defect can be rescued by HTT phosphorylation at S421.

The structure of the TFIIH p34 subunit reveals a von Willebrand factor A like fold.

RNA polymerase II dependent transcription and nucleotide excision repair are mediated by a multifaceted interplay of subunits within the general transcription factor II H (TFIIH). A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these vital cellular processes. The importance of this complex becomes further evident in the context of severe diseases like xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy, that arise from single point mutations in TFIIH subunits. Here we describe the structure of the p34 subunit of the TFIIH complex from the eukaryotic thermophilic fungus Chaetomium thermophilum. The structure revealed that p34 contains a von Willebrand Factor A (vWA) like domain, a fold which is generally known to be involved in protein-protein interactions. Within TFIIH p34 strongly interacts with p44, a positive regulator of the helicase XPD. Putative protein-protein interfaces are analyzed and possible binding sites for the p34-p44 interaction suggested.

Association between an alternative promoter polymorphism and sperm deformity rate is due to modulation of the expression of KATNAL1 transcripts in Chinese Holstein bulls.

Katanin p60 subunit A-like 1 (KATNAL1) is an ATPase that regulates Sertoli cell microtubule dynamics and sperm retention. We evaluated one novel splice variant and characterized the promoter and a functional single nucleotide polymorphism (SNP) of the bovine KATNAL1 gene to explore its expression pattern, possible regulatory mechanism and relationship with semen traits in Chinese Holstein bulls. A novel splice variant, KATNAL1 transcript variant 2 (KATNAL1-TV2) of the retained 68bp in intron 2, was identified by RT-PCR and compared with KATNAL1 transcript variant 1 (KATNAL1-TV1, NM 001192918.1) in various tissues. Bioinformatics analyses predicted that KATNAL1 transcription was regulated by two promoters: P1 in KATNAL1-TV1 and P2 in KATNAL1-TV2. Results of qRT-PCR revealed that KATNAL1-TV1 had higher expression than did KATNAL1-TV2 in testes of adult bulls (P<0.05). Promoter luciferase activity analysis suggested that the core sequences of P1 and P2 were mapped to the region of c.-575˜c.-180 and c.163-40˜c.333+59 respectively. One novel SNP (c.163-210T>C, ss836312085) located in intron 1 was found using sequence alignment. The SNP in P2 resulted in the presence of the DeltaE binding site, improving its basal promoter activity (P<0.05); and we observed a greater sperm deformity rate in bulls with the genotype CC than in those with the genotype TT (P<0.05), which indicated that different genotypes were associated with the bovine semen traits. Bioinformatics analysis of the KATNAL1 protein sequence predicted that the loss of the MIT domain in the KATNAL1-TV2 transcript resulted in protein dysfunction. These findings help us to understand that a functional SNP in P2 and subsequent triggering of expression diversity of KATNAL1 transcripts are likely to play an important role with regard to semen traits in bull breeding programs.

Nuclear translocation of IGF-1R via p150(Glued) and an importin-β/RanBP2-dependent pathway in cancer cells.

Mounting evidence has shown that the insulin-like growth factor-1 receptor (IGF-1R) has critical roles in cancer cell growth. This has prompted pharmacological companies to develop agents targeting the receptor. Surprisingly, clinical trials using specific IGF-1R antibodies have, however, revealed disappointing results. Further understanding of the role of IGF-1R in cancer cells is therefore necessary for development of efficient therapeutic strategies. Recently, we showed that IGF-1R is sumoylated and translocated into the cell nucleus where it activates gene transcription. Several other studies have confirmed our findings and it has been reported that nuclear IGF-1R (nIGF-1R) has prognostic and predictive impact in cancer. To increase the understanding of IGF-1R in cancer cells, we here present the first study that proposes a pathway by which IGF-1R translocates into the cell nucleus. We could demonstrate that IGF-1R first associates with the dynactin subunit p150(Glued), which transports the receptor to the nuclear pore complex, where it co-localizes with importin-β followed by association with RanBP2. Sumoylation of IGF-1R seems to be required for interaction with RanBP2, which in turn may serve as the SUMO E3 ligase. In the context of sumoylation, we provided evidence that it may favor nIGF-1R accumulation by increasing the stability of the receptor. Taken together, topographic and functional interactions between dynactin, importin-β and RanBP2 are involved in nuclear translocation of IGF-1R. Our results provide new understanding of IGF-1R in cancer, which in turn may contribute to development of new therapeutic strategies.

Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

SummaryCopper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease.

Up-regulation of CHAF1A, a poor prognostic factor, facilitates cell proliferation of colon cancer

Deregulation of chromatin assembly factor 1, p150 subunit A (CHAF1A) has recently been reported to be involved in the development of some cancer types. In this study, we identified that the frequency of positive CHAF1A staining in primary tumor mucosa (45.8%, 93 of 203 samples) was significantly elevated compared to that in paired normal mucosa (18.7%, 38 of 203 samples). The increased expression was strongly associated with cancer stage, tumor invasion, and histological grade. The five-year survival rate of patients with CHAF1A-positive tumors was remarkably lower than that of patients with CHAF1A-negative tumors. Colon cancer cells with CHAF1A knockdown exhibited decreased cell growth index, reduction in colony formation ability, elevated cell apoptosis rate as well as impaired colon tumorigenicity in nude mice. Hence, CHAF1A upregulation functions as a poor prognostic indicator of colon cancer, potentially contributing to its progression by mediating cancer cell proliferation.

Dynactin integrity depends upon direct binding of dynamitin to Arp1.

Dynactin is a multiprotein complex that works with cytoplasmic dynein and other motors to support a wide range of cell functions. It serves as an adaptor that binds both dynein and cargoes and enhances single-motor processivity. The dynactin subunit dynamitin (also known as p50) is believed to be integral to dynactin structure because free dynamitin displaces the dynein-binding p150(Glued) subunit from the cargo-binding Arp1 filament. We show here that the intrinsically disordered dynamitin N-terminus binds to Arp1 directly. When expressed in cells, dynamitin amino acids (AA) 1-87 causes complete release of endogenous dynamitin, p150, and p24 from dynactin, leaving behind Arp1 filaments carrying the remaining dynactin subunits (CapZ, p62, Arp11, p27, and p25). Tandem-affinity purification-tagged dynamitin AA 1-87 binds the Arp filament specifically, and binding studies with purified native Arp1 reveal that this fragment binds Arp1 directly. Neither CapZ nor the p27/p25 dimer contributes to interactions between dynamitin and the Arp filament. This work demonstrates for the first time that Arp1 can directly bind any protein besides another Arp and provides important new insight into the underpinnings of dynactin structure.

Proteolysis of the human DNA polymerase delta smallest subunit p12 by μ-calpain in calcium-triggered apoptotic HeLa cells.

Degradation of p12 subunit of human DNA polymerase delta (Pol δ) that results in an interconversion between Pol δ4 and Pol δ3 forms plays a significant role in response to replication stress or genotoxic agents triggered DNA damage. Also, the p12 is readily degraded by human calpain in vitro. However, little has been done for the investigation of its degree of participation in any of the more common apoptosis. Here, we first report that the p12 subunit is a substrate of μ-calpain. In calcium-triggered apoptotic HeLa cells, the p12 is degraded at 12 hours post-induction (hpi), restored thereafter by 24 hpi, and then depleted again after 36 hpi in a time-dependent manner while the other three subunits are not affected. It suggests a dual function of Pol δ by its interconversion between Pol δ4 and Pol δ3 that is involved in a novel unknown apoptosis mechanism. The proteolysis of p12 could be efficiently blocked by both calpain inhibitor ALLN and proteasome inhibitor MG132. In vitro pull down and co-immunoprecipitation assays show that the μ-calpain binds to p12 through the interaction of μ-calpain with Pol δ other three subunits, not p12 itself, and PCNA, implying that the proteolysis of p12 by μ-calpain might be through a Pol δ4/PCNA complex. The p12 cleavage sites by μ-calpain are further determined as the location within a 16-amino acids peptide 28-43 by in vitro cleavage assays. Thus, the p12/Pol δ is a target as a nuclear substrate of μ-calpain in a calcium-triggered apoptosis and appears to be a potential marker in the study of the chemotherapy of cancer therapies.

Evidence for the hypothesis that two forms of human DNA polymerase δ participate in DNA replication (925.2)

Eukaryotic DNA polymerase δ (Pol δ) is the polymerase primarily involved in lagging strand DNA synthesis. Human Pol δ is a heterotetramer (Pol δ4) composed of four subunits. The p12 subunit is degraded in response to DNA damage, resulting in the in vivo conversion of Pol δ4 to Pol δ3, the trimer lacking p12. Thus, Pol δ3 is the form that is involved in DNA repair. The hypothesis that Pol δ3 is also capable of performing lagging strand DNA synthesis was tested in a reconstituted system for Okazaki fragment processing using purified enzymes and model oligonucleotide substrates. In the combined Pol δ3/Fen1 reactions, the products formed by removal of the downstream blocking primer were almost exclusively mononucleotides, showing that the reaction proceeded by nick translation, and was not affected by Fen1 concentrations. With Pol δ4/Fen1, the products were 1‐10 nts at low Fen1 concentrations, showing that the reaction proceeded by the short flap pathway. As Fen1 concentrations were increased the product size was shifted to mono‐nucleotides, i.e.,. to a nick translation mode. These studies show that Pol δ3 is ideally suited for Okazaki fragment processing. In parallel studies, we have shown that p12 is degraded during the G1/S transition so that a mixture of Pol δ4 and Pol δ3 is present in S phase, under the control of CRL4Cdt2, which also regulates the degradation of the licensing factors Cdt1, Set8 and p21.Grant Funding Source: Supported by GM31973 and ES14737

Molecular Mechanisms Underlying the Nephroprotective Effects of PACAP in Diabetes

Diabetic nephropathy is the leading cause of end-stage renal failure and accounts for 30-40% of patients entering renal transplant programmes. The nephroprotective effects of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP38) against diabetes have been shown previously, but the molecular mechanisms responsible for these effects remain unknown. In the present study, we showed that PACAP treatment counteracted the diabetes-induced increase in the level of the proapoptotic pp38MAPK and cleaved caspase-3 and also decreased the p60 subunit of NFκB. The examined antiapoptotic factors, including pAkt and pERK1/2, showed a slight increase in the diabetic kidneys, while PACAP treatment resulted in a notable elevation of these proteins. PCR and Western blot revealed the downregulation of fibrotic markers, like collagen IV and TGF-β1 in the kidney. PACAP treatment resulted in increased expression of the antioxidant glutathione. We conclude that the nephroprotective effect of PACAP in diabetes is, at least partly, due to its antiapoptotic, antifibrotic and antioxidative effect in addition to the previously described antiinflammatory effect.

Virulence Factor NSs of Rift Valley Fever Virus Recruits the F-Box Protein FBXO3 To Degrade Subunit p62 of General Transcription Factor TFIIH

The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity.

New Folding Pattern of P150 and “Anntena” for Dynein Binding

Dynactin is known to be a regulator of cytoplasmic dynein and play a part in intracellular transport. Dynactin is a large complex composed of multiple subunits including p150, p50 and Arp1, and has a characteristic architecture. Sidearm domain of dynactin is essential for binding to dynein and microtubule, but the structural detail of the domain is poorly understood due to its intricate organization. Here, by electron microscopy of the nanogold-labeled or the truncated mutants of human dynactin complex, the location of each domain of the largest subunit p150 was identified. We rediscovered the filamentous structure at the very tip of sidearm as a newly named “antenna”, which is comprised of two anti-parallel coiled-coil structures and crucial for dynein-binding. Furthermore, the composition of the globular head which is important for microtubule-binding is also renewed as the complex of the two discontinuous regions. These findings provide the clues for understanding dynactin functions and its molecular mechanism, especially as a regulator of dynein.

Insights into Eukaryotic Primer Synthesis from Structures of the p48 Subunit of Human DNA Primase

DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8–10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal that eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique subdomain not found in the archaeal and bacterial primases. Calorimetry experiments reveal that Mn2+ but not Mg2+ significantly enhances the binding of nucleotide to primase, which correlates with higher catalytic efficiency in vitro. The structure of p48 with bound UTP and Mn2+ provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding alter nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions are not viable. Our results reveal that two residues (S160 and H166) in direct contact with the nucleotide were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.

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.