skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

We report the crystal structure of the middle domain of the Pob3 subunit (Pob3-M) of S. cerevisiae FACT (yFACT, facilitates chromatin transcription), which unexpectedly adopts an unusual double pleckstrin homology (PH) architecture. A mutation within a conserved surface cluster in this domain causes a defect in DNA replication that is suppressed by mutation of replication protein A (RPA). The nucleosome reorganizer yFACT therefore interacts in a physiologically important way with the central single-strand DNA (ssDNA) binding factor RPA to promote a step in DNA replication. Purified yFACT and RPA display a weak direct physical interaction, although the genetic suppression is not explained by simple changes in affinity between the purified proteins. Further genetic analysis suggests that coordinated function by yFACT and RPA is important during nucleosome deposition. These results support the model that the FACT family has an essential role in constructing nucleosomes during DNA replication, and suggest that RPA contributes to this process.

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
914318
Report Number(s):
BNL-78886-2007-JA
TRN: US200809%%173
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Mol. Cell; Journal Volume: 22
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; AFFINITY; ARCHITECTURE; CHROMATIN; CRYSTAL STRUCTURE; DEFECTS; DEPOSITION; DNA; DNA REPLICATION; GENETICS; MUTATIONS; NUCLEOSOMES; PROTEINS; TRANSCRIPTION; national synchrotron light source

Citation Formats

VanDemark,A., Blanksma, M., Ferris, E., Heroux, A., Hill, C., and Formosa, T.. The Structure of the yFACT Pob3-M Domain, Its Interaction with the DNA Replication Factor RPA, and a Potential Role in Nucleosome Deposition. United States: N. p., 2006. Web. doi:10.1016/j.molcel.2006.03.025.
VanDemark,A., Blanksma, M., Ferris, E., Heroux, A., Hill, C., & Formosa, T.. The Structure of the yFACT Pob3-M Domain, Its Interaction with the DNA Replication Factor RPA, and a Potential Role in Nucleosome Deposition. United States. doi:10.1016/j.molcel.2006.03.025.
VanDemark,A., Blanksma, M., Ferris, E., Heroux, A., Hill, C., and Formosa, T.. Sun . "The Structure of the yFACT Pob3-M Domain, Its Interaction with the DNA Replication Factor RPA, and a Potential Role in Nucleosome Deposition". United States. doi:10.1016/j.molcel.2006.03.025.
@article{osti_914318,
title = {The Structure of the yFACT Pob3-M Domain, Its Interaction with the DNA Replication Factor RPA, and a Potential Role in Nucleosome Deposition},
author = {VanDemark,A. and Blanksma, M. and Ferris, E. and Heroux, A. and Hill, C. and Formosa, T.},
abstractNote = {We report the crystal structure of the middle domain of the Pob3 subunit (Pob3-M) of S. cerevisiae FACT (yFACT, facilitates chromatin transcription), which unexpectedly adopts an unusual double pleckstrin homology (PH) architecture. A mutation within a conserved surface cluster in this domain causes a defect in DNA replication that is suppressed by mutation of replication protein A (RPA). The nucleosome reorganizer yFACT therefore interacts in a physiologically important way with the central single-strand DNA (ssDNA) binding factor RPA to promote a step in DNA replication. Purified yFACT and RPA display a weak direct physical interaction, although the genetic suppression is not explained by simple changes in affinity between the purified proteins. Further genetic analysis suggests that coordinated function by yFACT and RPA is important during nucleosome deposition. These results support the model that the FACT family has an essential role in constructing nucleosomes during DNA replication, and suggest that RPA contributes to this process.},
doi = {10.1016/j.molcel.2006.03.025},
journal = {Mol. Cell},
number = ,
volume = 22,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • Replication Protein A (RPA) is a heterotrimeric (subunits of 70-, 32- & 14-kDa) single-stranded DNA-binding protein that is required for DNA replication, recombination and repair. The 40 residue N-terminal domain of the 32-kDa subunit of RPA (RPA32) becomes phosphorylated during S-phase and after DNA damage. Recently it has been shown that phosphorylation or the addition of negative charges to this N-terminal phosphorylation domain modulates RPA-protein interactions and increases cell sensitivity to DNA damage (Braun et al in preparation). We found that addition of multiple negative charges to the N-terminal phosphorylation domain also caused a significant decrease in the ability ofmore » a mutant form of RPA to destabilize double-stranded DNA (dsDNA). Kinetic studies suggested that the addition of negative charges to the N-terminal phosphorylation domain caused defects in both complex formation (nucleation) and subsequent destabilization of dsDNA by RPA. We conclude that the N-terminal phosphorylation domain modulates RPA interactions with dsDNA. Similar changes in DNA interactions were observed with a mutant form of RPA in which the N-terminal domain of the 70-kDa subunit was deleted. This suggested a functional link between the N-terminal domains of the 70- and 32-kDa subunits of RPA. NMR experiments provided evidence for a direct interaction between the N-terminal domain of the 70-kDa subunit and the negatively charged N-terminal phosphorylation domain of RPA32. These findings suggest that phosphorylation causes a conformational change in the RPA complex that regulates RPA function.« less
  • Cited by 18
  • Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB's association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes inmore » χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.« less
  • ZEBRA, a transcription factor and DNA replication protein encoded by the Epstein-Barr virus (EBV) BZLF1 gene, plays indispensable roles in the EBV lytic cycle. We recently described the phenotypes of 46 single amino acid substitutions introduced into the DNA-recognition region of ZEBRA [Heston, L., El-Guindy, A., Countryman, J., Dela Cruz, C., Delecluse, H.J., and Miller, G. 2006]. The 27 DNA-binding-proficient mutants exhibited distinct defects in their ability to activate expression of the kinetic classes of viral genes. Four phenotypic variants could be discerned: wild-type, defective at activating Rta, defective at activating early genes, and defective at activating late genes. Heremore » we analyze the distribution of ZEBRA within the nucleus and the localization of EA-D (the viral DNA polymerase processivity factor), an indicator of the development of replication compartments, in representatives of each phenotypic group. Plasmids encoding wild-type (WT) and mutant ZEBRA were transfected into 293 cells containing EBV-bacmids. WT ZEBRA protein was diffusely and smoothly distributed throughout the nucleus, sparing nucleoli, and partially recruited to globular replication compartments. EA-D induced by WT ZEBRA was present diffusely in some cells and concentrated in globular replication compartments in other cells. The distribution of ZEBRA and EA-D proteins was identical to WT following transfection of K188R, a mutant with a conservative change. The distribution of S186A mutant ZEBRA protein, defective for activation of Rta and EA-D, was identical to WT, except that the mutant ZEBRA was never found in globular compartments. Co-expression of Rta with S186A mutant rescued diffuse EA-D but not globular replication compartments. The most striking observation was that several mutant ZEBRA proteins defective in activating EA-D (R179A, K181A and A185V) and defective in activating lytic viral DNA replication and late genes (Y180E and K188A) were localized to numerous punctate foci. The speckled appearance of R179A and Y180E was more regular and clearly defined in EBV-positive than in EBV-negative 293 cells. The Y180E late-mutant induced EA-D, but prevented EA-D from localizing to globular replication compartments. These results show that individual amino acids within the basic domain influence localization of the ZEBRA protein and its capacity to induce EA-D to become located in mature viral replication compartments. Furthermore, these mutant ZEBRA proteins delineate several stages in the processes of nuclear re-organization which accompany lytic EBV replication.« less