Abstract P-28: Structural Dynamics of DNA-Associated Chaperon Facilitates Chromatine Transcription

• DOI: 10.21103/ijbm.11.suppl_1.p28
• Publication Date: 2021-10-01T17:26:55Z
• Authors: Olesya Volokh; Anastasia Sivkina; Maria Karlova; Elena Kotova; Vasily Studitsky; Olga Sokolova
• Keywords: Transcription; Cryo-Electron Microscopy

Background: Histone chaperon FACT (“FAcilitates Chromatin Transcription”) is a multifunctional and conserved eukaryotic protein involved in DNA transcription, replication repair; which can reversibly unfold nucleosomes presence of ATP. necessary for the viability growth breast tumor cells meanwhile normal it be knocked out without loss vitality. Human (hFACT) target promising anticancer drug curaxins, causes trapping chromatin cancer destabilizes nucleosome. The nucleosome-unfolding activity an important function hFACT vivo; however, mechanism FACT-dependent nucleosome unfolding remains unknown. Methods: Here, we studied negative stained structure using single particle electron microscopy JEOL 2100 TEM. Micrographs were captured with 25k magnification, 4.1 Å pixel size. EM images pre-processing particles collection performed EMAN2.3, followed by 2D-particles analysis RELION2.0. Final 2D-classes included ~70 000 images. Results: Based on 2D-classess data evaluated several states reflecting its conformational flexibility: “closed” complex characterized four domains localized close to each other forming compact structure; “intermediate” state represented classes identified three having more disordered fourth domain, “open” complex, almost linear structure. are present comparable amounts significantly outnumber state. It has been shown that connected through flexible linkers SPT16 SSRP1 dimerization (DDs) form “joint”-like connection between two subunits. In “сlosed” conformation, DNA-binding surface covered C-terminal middle (MDs). N-terminal domain (NTD) was not resolved previously, but best candidate forth clearly visible only conformation hFACT, based dimensions longest linker length. Conclusion: We propose during conversion complexes NTD moving away from subunits leading formation first intermediate poorly or resolved, while less mobile DDs MDs maintain site still protected CTDs. SPT16/SSRP1 structure, unmasking sites making them accessible interaction