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Structure of the quaternary complex of histone H3-H4 heterodimer with chaperone ASF1 and the replicative helicase subunit MCM2.

Wang H, Wang M, Yang N, Xu RM - Protein Cell (2015)

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

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The building block of eukaryotic chromatin is the nucleosome core particle (NCP), which is consisted of ~146 bps of DNA wrapped around an octamer of core histones... A tetramer of histone H3 and H4 and two H2A-H2B dimers form the histone octamer (Kornberg, ; Luger et al., ; Thomas and Kornberg, )... During DNA replication, nucleosome disassembly and reassembly occurs at the replication fork, and histone chaperons CAF-1 and ASF1 are principally responsible for the deposition of histones H3 and H4 onto replicated DNA (Kaufman et al., ; Tyler et al., )... The projection of the C-terminal segment of ASF1 is largely determined by the position of the immediately N-terminal strand β9, which is stabilized by antiparallel pairing with the C-terminal β-strand (βC) of histone H4 (Fig.  1B)... It is interesting to note that while βC is positioned similarly in our quaternary complex and the ASF1-H3-H4 ternary complex, it contrasts sharply with that found in the (MCM2-H3-H4)2 complex (Fig.  1E)... Superposition of our structure with the NCP structure (PDB code: 2CV5, shows in gray) via the H3-H4 heterodimer shows that (1) ASF1 occupies a position blocking the dimerization interface of histone H3, thus preventing the formation of a (H3-H4)2 tetramer (Fig.  2A, Region 1); (2) MCM2 occludes the binding of histone H2B to histone H4 through its α2 helix, which impedes the association of H2A-H2B heterodimers with (H3-H4)2 tetramer to form an octamer (Fig.  2A, Region 2); (3) both the binding of MCM2 to the positively charged surface of the H3-H4 heterodimer and the protrusion of the C-terminal α2 helix of ASF1 would obstruct the wrapping of DNA in NCP... Exactly how ASF1 is recruited to the replication or damage foci remains poorly understood, except that ASF1 is known to directly interact with the CAF-1 complex (Mello et al., )... A recent result also showed the association of MCM complex with ASF1 in U2OS cells (Drissi et al., )... A summary of possible cellular functions of the interactions among MCM2, ASF1 and histones H3 and H4 is depicted in Fig.  2B, and the structural basis for their interactions learned here should help the dissection of their functions in chromatin biology... N.Y. is also supported by the Youth Innovation Promotion Association of CAS... All authors declare that they have no conflict of interest... This article does not contain any studies with human or animal subjects performed by any of the authors... Supplementary material 1 (PDF 249 kb)

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Structure of the MCM2-ASF1-H3-H4 complex. (A) Schematic diagram showing the truncated fragments of the four proteins. MCM2, ASF1, H3 and H4 are shown in yellow, green, cyan and magenta respectively (color coded the same in all figures). Disordered regions in the structure are shown in gray. An image of the coomassie-stained SDS-PAGE gel of the purified complex showing apparent stoichiometry of the four proteins. MCM2 appears to have an anomalous SDS-PAGE migration profile, as a Mass spectrometric measurement indicates a molecular weight 10,774 Da (data not shown). (B) A ribbon diagram showing the overall structure of the quaternary complex. (C) Interactions between MCM2 and H3-H4 heterodimer. The structure of MCM2 from the ternary MCM2-H3-H4 structure (PDB code: 4UUZ, shown in gray) is superimposed for comparison. Three panels indicate N-terminal, middle and C-terminal binding regions of MCM2. Residues involved in intermolecular interactions are shown in a stick model (carbon, yellow, magenta and cyan; nitrogen, blue; oxygen, red). Dashed lines indicate intermolecular hydrogen bonds. (D) Interactions between ASF1 and histone H3. Superposition of ASF1 and H3 from our quaternary structure and that from the ternary ASF1-H3-H4 structure (PDB code: 2HUE, shown in gray). C-terminal ends of ASF1 in the two structures and α2 MCM2 are highlighted inside the red circle. (E) Interactions between ASF1 and histone H4. The βC strand of H4 in the MCM2-H3-H4 ternary structure and in a human nucleosome structure (shown in gray and magenta, respectively) are aligned with that in the quaternary structure. Histone H2A in the nucleosome structure are shown in red. (F) Direct interactions between MCM2 and ASF1
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Fig1: Structure of the MCM2-ASF1-H3-H4 complex. (A) Schematic diagram showing the truncated fragments of the four proteins. MCM2, ASF1, H3 and H4 are shown in yellow, green, cyan and magenta respectively (color coded the same in all figures). Disordered regions in the structure are shown in gray. An image of the coomassie-stained SDS-PAGE gel of the purified complex showing apparent stoichiometry of the four proteins. MCM2 appears to have an anomalous SDS-PAGE migration profile, as a Mass spectrometric measurement indicates a molecular weight 10,774 Da (data not shown). (B) A ribbon diagram showing the overall structure of the quaternary complex. (C) Interactions between MCM2 and H3-H4 heterodimer. The structure of MCM2 from the ternary MCM2-H3-H4 structure (PDB code: 4UUZ, shown in gray) is superimposed for comparison. Three panels indicate N-terminal, middle and C-terminal binding regions of MCM2. Residues involved in intermolecular interactions are shown in a stick model (carbon, yellow, magenta and cyan; nitrogen, blue; oxygen, red). Dashed lines indicate intermolecular hydrogen bonds. (D) Interactions between ASF1 and histone H3. Superposition of ASF1 and H3 from our quaternary structure and that from the ternary ASF1-H3-H4 structure (PDB code: 2HUE, shown in gray). C-terminal ends of ASF1 in the two structures and α2 MCM2 are highlighted inside the red circle. (E) Interactions between ASF1 and histone H4. The βC strand of H4 in the MCM2-H3-H4 ternary structure and in a human nucleosome structure (shown in gray and magenta, respectively) are aligned with that in the quaternary structure. Histone H2A in the nucleosome structure are shown in red. (F) Direct interactions between MCM2 and ASF1

Mentions: To answer these questions, we embarked on the characterization and structure determination of a quaternary complex of MCM2, ASF1 and histones H3 and H4. First, we find that they form an apparent 1:1:1:1 complex. The N-terminal domain of human MCM2 (aa 63–154), the globular domain of ASF1a (aa 1–157) and full-length human histones H3 and H4 were expressed in E. coli. A stable complex of the four proteins was obtained at a salt concentration of 0.5 mol/L NaCl (Fig. 1A). The tetrameric complex eluded from a Superdex 200 10/300 GL size exclusion column (GE Healthcare) at an elution volume of 14.38 mL (Fig. S1), corresponding to an apparent molecular weight of about 60 kDa, which is compatible with a stoichiometry of 1:1:1:1 of the four components. We then crystallized and solved a 3.5-Å structure of the quaternary complex, and the structure was solved by molecular replacement (see Supplemental Material for details). There are six MCM2-ASF1-H3-H4 tetramers in one asymmetric unit, the inter-tetramer contacts appear to be non-physiological, and we will concentrate our analyses on one tetramer henceforth.Figure 1


Structure of the quaternary complex of histone H3-H4 heterodimer with chaperone ASF1 and the replicative helicase subunit MCM2.

Wang H, Wang M, Yang N, Xu RM - Protein Cell (2015)

Structure of the MCM2-ASF1-H3-H4 complex. (A) Schematic diagram showing the truncated fragments of the four proteins. MCM2, ASF1, H3 and H4 are shown in yellow, green, cyan and magenta respectively (color coded the same in all figures). Disordered regions in the structure are shown in gray. An image of the coomassie-stained SDS-PAGE gel of the purified complex showing apparent stoichiometry of the four proteins. MCM2 appears to have an anomalous SDS-PAGE migration profile, as a Mass spectrometric measurement indicates a molecular weight 10,774 Da (data not shown). (B) A ribbon diagram showing the overall structure of the quaternary complex. (C) Interactions between MCM2 and H3-H4 heterodimer. The structure of MCM2 from the ternary MCM2-H3-H4 structure (PDB code: 4UUZ, shown in gray) is superimposed for comparison. Three panels indicate N-terminal, middle and C-terminal binding regions of MCM2. Residues involved in intermolecular interactions are shown in a stick model (carbon, yellow, magenta and cyan; nitrogen, blue; oxygen, red). Dashed lines indicate intermolecular hydrogen bonds. (D) Interactions between ASF1 and histone H3. Superposition of ASF1 and H3 from our quaternary structure and that from the ternary ASF1-H3-H4 structure (PDB code: 2HUE, shown in gray). C-terminal ends of ASF1 in the two structures and α2 MCM2 are highlighted inside the red circle. (E) Interactions between ASF1 and histone H4. The βC strand of H4 in the MCM2-H3-H4 ternary structure and in a human nucleosome structure (shown in gray and magenta, respectively) are aligned with that in the quaternary structure. Histone H2A in the nucleosome structure are shown in red. (F) Direct interactions between MCM2 and ASF1
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Fig1: Structure of the MCM2-ASF1-H3-H4 complex. (A) Schematic diagram showing the truncated fragments of the four proteins. MCM2, ASF1, H3 and H4 are shown in yellow, green, cyan and magenta respectively (color coded the same in all figures). Disordered regions in the structure are shown in gray. An image of the coomassie-stained SDS-PAGE gel of the purified complex showing apparent stoichiometry of the four proteins. MCM2 appears to have an anomalous SDS-PAGE migration profile, as a Mass spectrometric measurement indicates a molecular weight 10,774 Da (data not shown). (B) A ribbon diagram showing the overall structure of the quaternary complex. (C) Interactions between MCM2 and H3-H4 heterodimer. The structure of MCM2 from the ternary MCM2-H3-H4 structure (PDB code: 4UUZ, shown in gray) is superimposed for comparison. Three panels indicate N-terminal, middle and C-terminal binding regions of MCM2. Residues involved in intermolecular interactions are shown in a stick model (carbon, yellow, magenta and cyan; nitrogen, blue; oxygen, red). Dashed lines indicate intermolecular hydrogen bonds. (D) Interactions between ASF1 and histone H3. Superposition of ASF1 and H3 from our quaternary structure and that from the ternary ASF1-H3-H4 structure (PDB code: 2HUE, shown in gray). C-terminal ends of ASF1 in the two structures and α2 MCM2 are highlighted inside the red circle. (E) Interactions between ASF1 and histone H4. The βC strand of H4 in the MCM2-H3-H4 ternary structure and in a human nucleosome structure (shown in gray and magenta, respectively) are aligned with that in the quaternary structure. Histone H2A in the nucleosome structure are shown in red. (F) Direct interactions between MCM2 and ASF1
Mentions: To answer these questions, we embarked on the characterization and structure determination of a quaternary complex of MCM2, ASF1 and histones H3 and H4. First, we find that they form an apparent 1:1:1:1 complex. The N-terminal domain of human MCM2 (aa 63–154), the globular domain of ASF1a (aa 1–157) and full-length human histones H3 and H4 were expressed in E. coli. A stable complex of the four proteins was obtained at a salt concentration of 0.5 mol/L NaCl (Fig. 1A). The tetrameric complex eluded from a Superdex 200 10/300 GL size exclusion column (GE Healthcare) at an elution volume of 14.38 mL (Fig. S1), corresponding to an apparent molecular weight of about 60 kDa, which is compatible with a stoichiometry of 1:1:1:1 of the four components. We then crystallized and solved a 3.5-Å structure of the quaternary complex, and the structure was solved by molecular replacement (see Supplemental Material for details). There are six MCM2-ASF1-H3-H4 tetramers in one asymmetric unit, the inter-tetramer contacts appear to be non-physiological, and we will concentrate our analyses on one tetramer henceforth.Figure 1

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

The building block of eukaryotic chromatin is the nucleosome core particle (NCP), which is consisted of ~146 bps of DNA wrapped around an octamer of core histones... A tetramer of histone H3 and H4 and two H2A-H2B dimers form the histone octamer (Kornberg, ; Luger et al., ; Thomas and Kornberg, )... During DNA replication, nucleosome disassembly and reassembly occurs at the replication fork, and histone chaperons CAF-1 and ASF1 are principally responsible for the deposition of histones H3 and H4 onto replicated DNA (Kaufman et al., ; Tyler et al., )... The projection of the C-terminal segment of ASF1 is largely determined by the position of the immediately N-terminal strand β9, which is stabilized by antiparallel pairing with the C-terminal β-strand (βC) of histone H4 (Fig.  1B)... It is interesting to note that while βC is positioned similarly in our quaternary complex and the ASF1-H3-H4 ternary complex, it contrasts sharply with that found in the (MCM2-H3-H4)2 complex (Fig.  1E)... Superposition of our structure with the NCP structure (PDB code: 2CV5, shows in gray) via the H3-H4 heterodimer shows that (1) ASF1 occupies a position blocking the dimerization interface of histone H3, thus preventing the formation of a (H3-H4)2 tetramer (Fig.  2A, Region 1); (2) MCM2 occludes the binding of histone H2B to histone H4 through its α2 helix, which impedes the association of H2A-H2B heterodimers with (H3-H4)2 tetramer to form an octamer (Fig.  2A, Region 2); (3) both the binding of MCM2 to the positively charged surface of the H3-H4 heterodimer and the protrusion of the C-terminal α2 helix of ASF1 would obstruct the wrapping of DNA in NCP... Exactly how ASF1 is recruited to the replication or damage foci remains poorly understood, except that ASF1 is known to directly interact with the CAF-1 complex (Mello et al., )... A recent result also showed the association of MCM complex with ASF1 in U2OS cells (Drissi et al., )... A summary of possible cellular functions of the interactions among MCM2, ASF1 and histones H3 and H4 is depicted in Fig.  2B, and the structural basis for their interactions learned here should help the dissection of their functions in chromatin biology... N.Y. is also supported by the Youth Innovation Promotion Association of CAS... All authors declare that they have no conflict of interest... This article does not contain any studies with human or animal subjects performed by any of the authors... Supplementary material 1 (PDF 249 kb)

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