The Structure of the yFACT Pob3-M Domain, Its Interaction with the DNA Replication Factor RPA, and a Potential Role in Nucleosome Deposition

VanDemark, Andrew P.; Blanksma, Mary; Ferris, Elliott; Héroux, A.; Hill, Christopher P.; Formosa, Tim

doi:10.1016/j.molcel.2006.03.025

Cited by 129 publications

(194 citation statements)

References 75 publications

(78 reference statements)

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“…In fact, recent findings indicate that the budding yeast N-terminal domain (Spt16-NTD) and the middle domain of Pob3 (Pob3-M) may mediate partially redundant functions and account for the viability of either single mutant (29). Indeed, both Spt16-NTD and the middle domain of Pob3 genetically interact with histones, and pob3-M spt16-NTD double mutants display synthetic defects (24,29).…”

Section: Discussionmentioning

confidence: 99%

“…Structural information on FACT exists for the HMG-like module Nhp6A from S. cerevisiae (23), which binds DNA, and for the middle domain of S. cerevisiae Pob3 (24), which resembles a double PH-like fold and interacts with replication protein A. The highly conserved Spt16 consists of three domains: an acidic segment at the C terminus (10) that is required for binding histones H2A-H2B in vitro (20) and resembles the acidic domains of Nap1, nucleolin, and Asf1 (25)(26)(27); two central domains, one of which interacts with Pob3 (21,24); and an N-terminal region of Ϸ450 residues (Spt16-N) showing homology with aminopeptidases (28). We were intrigued by the presence of a peptidase-like domain within this essential and conserved histone chaperone [supporting information (SI) Fig.…”

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confidence: 99%

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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.

130

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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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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.

130

141

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“…Hence, prediction of new PH‐like domains may identify residues that are functionally important for intracellular traffic. Since the initial discovery of classical PH domains, newly solved PH‐like structures have unexpectedly been found, both early on,17 and at least 10 times since 18, 19, 20, 21, 22, 23, 24, 25, 26, 27. Each of these discoveries added a new family of PH‐like domains to a growing PH‐like clan 28.…”

Section: Introductionmentioning

confidence: 99%

Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains

Fidler¹,

Murphy²,

Courtis³

et al. 2016

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Advances in membrane cell biology are hampered by the relatively high proportion of proteins with no known function. Such proteins are largely or entirely devoid of structurally significant domain annotations. Structural bioinformaticians have developed profile‐profile tools such as HHsearch (online version called HHpred), which can detect remote homologies that are missed by tools used to annotate databases. Here we have applied HHsearch to study a single structural fold in a single model organism as proof of principle. In the entire clan of protein domains sharing the pleckstrin homology domain fold in yeast, systematic application of HHsearch accurately identified known PH‐like domains. It also predicted 16 new domains in 13 yeast proteins many of which are implicated in intracellular traffic. One of these was Vps13p, where we confirmed the functional importance of the predicted PH‐like domain. Even though such predictions require considerable work to be corroborated, they are useful first steps. HHsearch should be applied more widely, particularly across entire proteomes of model organisms, to significantly improve database annotations.

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“…The FACT complex has been shown to interact with multiple fork components, including the Mcm2 -7 helicase complex and replication protein A (RPA) (Gambus et al 2006;Tan et al 2006;VanDemark et al 2006). Thus, FACT is well positioned to accept evicted H2A-H2B dimers and perhaps cooperate with NAP1 for their subsequent reassembly.…”

Section: Replication-independent Deposition Of Histone Variants H33 mentioning

confidence: 99%