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Updated: Sep 24, 2025
Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A
Published on: June 10, 2020
Biochemical and crystallization analysis of the CENP-SX-DNA complex
1Department of Biological Science and Technology, Faculty of Advanced Engineering, Tokyo University of Science (TUS), 6-3-1 Niijyuku, Katsushika-ku, Tokyo 125-8585, Japan.
Abstract:
The CENP-SX (MHF) complex is a conserved histone-fold protein complex that is involved in chromosome segregation and DNA repair. It can bind to DNA on its own as well as in complex with other proteins such as CENP-TW and FANCM to recognize specific substrates. CENP-SX binds nonspecifically to dsDNA, similar to other histone-fold proteins. Several low-resolution structures of CENP-SX in complex with DNA are known, but a high-resolution structure is still lacking. The DNA-binding properties of CENP-SX and FANCM-CENP-SX complexes with various lengths of dsDNA were compared and the band-shift patterns and migration positions were found to differ. To confirm the DNA-binding properties in detail, CENP-SX-DNA and FANCM-CENP-SX-DNA complexes were crystallized. Analysis of the crystals revealed that they all contained the CENP-SX-DNA complex, irrespective of the complex that was used in crystallization. Detailed diffraction data analyses revealed that there were two types of crystal with different space groups, P21 and C2, where the volume of the P21 asymmetric unit is twice as large as that of the C2 asymmetric unit. Analysis of the self-rotation function revealed the presence of twofold and fourfold symmetry in both crystals. This suggests that there may be multiple molecules of CENP-SX and DNA within the asymmetric unit with respective symmetry. Structure determination of the present crystals should reveal details of the DNA-binding properties of CENP-SX.
Insights
The CENP-SX complex, crucial for chromosome segregation and DNA repair, binds DNA. Crystallization and analysis revealed insights into its DNA-binding properties, paving the way for high-resolution structural determination.
Area of Science:
- Molecular Biology
- Structural Biology
- Genetics
Background:
- The CENP-SX (MHF) complex is a conserved histone-fold protein essential for chromosome segregation and DNA repair.
- It interacts with DNA independently and with other proteins like CENP-TW and FANCM to identify specific DNA substrates.
- While low-resolution structures exist, a high-resolution structure of CENP-SX bound to DNA is currently lacking.
Purpose of the Study:
- To investigate and detail the DNA-binding properties of the CENP-SX complex.
- To compare the DNA-binding characteristics of CENP-SX alone versus in complex with FANCM.
- To obtain crystals suitable for high-resolution structure determination of CENP-SX-DNA complexes.
Main Methods:
- Comparative analysis of DNA-binding properties using band-shift assays with varying dsDNA lengths for CENP-SX and FANCM-CENP-SX complexes.
- Crystallization of CENP-SX-DNA and FANCM-CENP-SX-DNA complexes.
- X-ray diffraction analysis of obtained crystals, including space group determination and self-rotation function analysis.
Main Results:
- DNA-binding properties differed between CENP-SX and FANCM-CENP-SX complexes, as indicated by distinct band-shift patterns and migration positions.
- Crystallization efforts yielded crystals containing the CENP-SX-DNA complex, regardless of the initial complex used.
- Two crystal forms (P21 and C2 space groups) were identified, suggesting potential multi-molecular arrangements within the asymmetric unit due to observed symmetries.
Conclusions:
- The study successfully generated crystals of CENP-SX-DNA complexes suitable for high-resolution structural analysis.
- The findings suggest the presence of symmetry and potential multi-protein-DNA interactions within the crystal structures.
- Further structure determination is expected to elucidate the detailed DNA-binding mechanisms of CENP-SX.
