A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs

Okuhara, Koji; Ohta, Kunihiro; Seo, Hidetaka; Shioda, Masaki; Yamada, Tsuneo; Tanaka, Yasuhiro; Dohmae, Naoshi; Seyama, Yousuke; Shibata, Takehiko; Murofushi, Hiromu

doi:10.1016/s0960-9822(99)80160-2

Cited by 98 publications

(107 citation statements)

References 43 publications

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“…Western Blotting Assay-Isolation of the chromatin fraction and Western blotting were performed as previously described (36) using antibody against MCM2/4 (a kind gift from Yukio Ishimi, Ibaraki University), Spt16 (29). DNA polymerase ␣ (a kind gift from Fumiko Hirose, University of Hyogo), RPA32 (Cell Signaling), histone H3 (Abcam), histone H2B (Upstate), phospho-Chk1 (Ser 345 ) (Cell Signaling), ␣-tubulin or FLAG-M2 (Sigma), followed by horseradish peroxidase-conjugated anti-rabbit, anti-rat, or anti-mouse IgG secondary antibody (Cell Signaling).…”

Section: Methodsmentioning

confidence: 99%

The Histone Chaperone Facilitates Chromatin Transcription (FACT) Protein Maintains Normal Replication Fork Rates

Abe

Sugimura

Hosono

et al. 2011

Journal of Biological Chemistry

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Ordered nucleosome disassembly and reassembly are required for eukaryotic DNA replication. The facilitates chromatin transcription (FACT) complex, a histone chaperone comprising Spt16 and SSRP1, is involved in DNA replication as well as transcription. FACT associates with the MCM helicase, which is involved in DNA replication initiation and elongation. Although the FACT-MCM complex is reported to regulate DNA replication initiation, its functional role in DNA replication elongation remains elusive. To elucidate the functional role of FACT in replication fork progression during DNA elongation in the cells, we generated and analyzed conditional SSRP1 gene knock-out chicken (Gallus gallus) DT40 cells. SSRP1-depleted cells ceased to grow and exhibited a delay in S-phase cell cycle progression, although SSRP1 depletion did not affect the level of chromatin-bound DNA polymerase ␣ or nucleosome reassembly on daughter strands. The tracking length of newly synthesized DNA, but not origin firing, was reduced in SSRP1-depleted cells, suggesting that the S-phase cell cycle delay is mainly due to the inhibition of replication fork progression rather than to defects in the initiation of DNA replication in these cells. We discuss the mechanisms of how FACT promotes replication fork progression in the cells.

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Section: Methodsmentioning

confidence: 99%

The Histone Chaperone Facilitates Chromatin Transcription (FACT) Protein Maintains Normal Replication Fork Rates

Abe

Sugimura

Hosono

et al. 2011

Journal of Biological Chemistry

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“…FACT is a highly conserved complex (2)(3)(4)(5). In fungi, SSRP1 is largely encoded by POB3, whereas NHP6 encodes the archetypal HMG-box of SSRP1.…”

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The FACT Spt16 “peptidase” domain is a histone H3–H4 binding module

Stuwe

Hothorn

Lejeune

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

127

141

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The FACT complex is a conserved cofactor for RNA polymerase II elongation through nucleosomes. FACT bears histone chaperone activity and contributes to chromatin integrity. However, the molecular mechanisms behind FACT function remain elusive. Here we report biochemical, structural, and mutational analyses that identify the peptidase homology domain of the Schizosaccharomyces pombe FACT large subunit Spt16 (Spt16-N) as a binding module for histones H3 and H4. The 2.1-Å crystal structure of Spt16-N reveals an aminopeptidase P fold whose enzymatic activity has been lost. Instead, the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails. Mutations within a conserved surface pocket in Spt16-N or posttranslational modification of the histone H4 tail reduce interaction in vitro, whereas the globular domains of H3-H4 and the H3 tail bind distinct Spt16-N surfaces. Our analysis suggests that the N-terminal domain of Spt16 may add to the known H2A-H2B chaperone activity of FACT by including a H3-H4 tail and H3-H4 core binding function mediated by the N terminus of Spt16. We suggest that these interactions may aid FACT-mediated nucleosome reorganization events.histone chaperone ͉ histone modifications ͉ protein evolution ͉ site-directed mutagenesis ͉ transcription N ucleosomes create a natural barrier to RNA polymerase II (Pol II) progression. Transcription of histone-wrapped DNA thus requires factors that promote nucleosome remodeling, such as the histone chaperone FACT (facilitates chromatin transcription), which in human cells purifies as a heterodimer of Spt16 and SSRP1 (1, 2).FACT is a highly conserved complex (2-5). In fungi, SSRP1 is largely encoded by POB3, whereas NHP6 encodes the archetypal HMG-box of SSRP1. The gene for the Spt16 subunit is essential in Saccharomyces cerevisiae and Schizosaccharomyces pombe, probably reflecting important chromatin-related functions in transcription, replication, and DNA repair (4, 6-9). Genetic screens identified Spt16 as a factor whose mutation restores the expression of a Ty1 transposon-silenced reporter gene by promoting its cryptic transcription, known as a suppressor of Ty1 phenotype, [Spt Ϫ ] (4, 7, 10, 11). Several POB3 alleles genes display [Spt Ϫ ] phenotypes (12), indicating that the maintenance of correct chromatin structure involves Pol II cofactors such as FACT (13). Indeed, FACT acts as a coactivator of transcriptional initiation and elongation (14), and many Spt16 alleles display genetic interactions with basal transcription factors. Furthermore, FACT subunits biochemically interact with the Pol II elongation complex Paf1 (15), bind the coding region of transcribed Pol II genes, and are recruited to inducible genes upon activation (16-19).FACT's biological roles in transcription (and replication) may stem from its histone chaperone activity (20,21). Histone chaperones stimulate reactions involving the transfer of histones (22), thereby mediating chromatin reorganiz...

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“…Nhp6 supports the function of Spt16-Pob3, a heterodimer implicated in both DNA replication and regulation of transcription (10 -12). Spt16 and Pob3 contain most of the sequences found in the components of the human FACT and frog DUF complexes (13,14), but Pob3 lacks the HMG box motif found in the otherwise homologous FACT subunit SSRP1 (12,15,16). Nhp6 appears to provide HMG box function for Spt16-Pob3 both in vivo and in vitro as cells lacking Nhp6 cannot tolerate mutations in SPT16 or POB3 (11,17), and Spt16-Pob3 can only bind to and reorganize nucleosomes in vitro if Nhp6 is present (11).…”

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confidence: 99%

Multiple Nhp6 Molecules Are Required to Recruit Spt16-Pob3 to Form yFACT Complexes and to Reorganize Nucleosomes

Ruone

Rhoades

Formosa

2003

Journal of Biological Chemistry

108

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The Saccharomyces cerevisiae Nhp6 protein contains a DNA-binding motif that is similar to those found in the high mobility group B family of chromatin proteins. Nhp6 bound to nucleosomes and made at least two changes in them: the nucleosomal DNA became more sensitive to DNase I at specific sites, and the nucleosomes became competent to bind Spt16-Pob3 to form yFACT⅐nucleosome complexes. Both changes occurred at similar concentrations of Nhp6, suggesting that they reflect the same structural reorganization of the nucleosome. Nucleosomes have multiple binding sites for Nhp6, and structural reorganization was associated with a concentration of Nhp6 about 10-fold higher than that needed for simple binding. We propose that the coordinated action of multiple Nhp6 molecules is required to convert nucleosomes to an alternative form as the first step in a two-step reorganization of nucleosomes with the second step being dependent on Spt16-Pob3. The presence of linker DNA had only subtle effects on these processes, indicating that both Nhp6 and yFACT act on core nucleosome structure rather than on the interaction between nucleosomes and adjacent DNA. These results suggest that Nhp6 and the related high mobility group B proteins may have a general role in promoting rearrangements of chromatin by initiating the destabilization of core nucleosomal structure.

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A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs

Abstract: We propose that DUF plays a role in DNA replication in Xenopus egg extracts.

Cited by 98 publications

References 43 publications

The Histone Chaperone Facilitates Chromatin Transcription (FACT) Protein Maintains Normal Replication Fork Rates

The Histone Chaperone Facilitates Chromatin Transcription (FACT) Protein Maintains Normal Replication Fork Rates

The FACT Spt16 “peptidase” domain is a histone H3–H4 binding module

Multiple Nhp6 Molecules Are Required to Recruit Spt16-Pob3 to Form yFACT Complexes and to Reorganize Nucleosomes

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