Structural elucidation of modular regulation of the Swi2/Snf2 ATPases Mot1 and Ino80

Woike, Stephan Wilhelm (2022): Structural elucidation of modular regulation of the Swi2/Snf2 ATPases Mot1 and Ino80. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

In eukaryotes, chromatin – the DNA packaged by nucleosomes and other bound proteins - is constantly reshaped by energy-dependent processes that facilitate accessibility of DNA for the replication, repair and transcription machinery. Swi2/Snf2 helicases translate energy derived from ATP-hydrolysis into DNA minor groove translocation resulting in either tracing DNA or pumping or pulling it to disrupt protein:DNA interactions, termed “chromatin remodeling”. The transcription regulator Mot1 is a single-subunit Swi2/Snf2 ATPase that removes TBP from the TATA box at the DNA promoter, thus recycling it and enabling a redistribution to other promoters. Despite a wealth of biochemistry, the chemo-mechanical details of the TBP removal were unknown. In the first publication, we present the crystal structure of near full-length Mot1 in an autoinhibited resting state. This allowed insight into the interaction between N-terminal HEATrepeat arch and C-terminal ATPase in nucleotide-free state. In the second publication, we employed cryogenic electron microscopy (cryo-EM) to determine five structures of Mot1 bound to its TBP:DNA substrate with different ATP analogues. We could therefore dissect the stepwise dissociation of TBP from DNA in molecular detail and analyze the structure and function of the outermost C-terminal “bridge” element as an allosteric regulator of the remodeling activity of Mot1. Ultimately, we arrived at a model that involves a short-range, non-processive DNA translocation by Mot1, including bending and rotation of the DNA. This is in contrast to the processive DNA translocation of nucleosome remodelers, usually multi-subunit complex molecular machines that pump DNA around the histone octamer and thus slide nucleosomes and some even facilitate histone ejection and variant exchange. The resulting spaced nucleosome arrays and nucleosome-free regions are a prerequisite for DNA replication, repair and transcription. The INO80 complex is such a mega-Dalton multi-subunit nucleosome remodeler. In the third publication, we investigated the structural basis of INO80’s allosteric regulation by the so-called “A-module”. The A-module consists of nuclear actin in complex with actin-related proteins bound to a lever that feeds back to the motor ATPase. Although it is known that the Amodule binds to extranucleosomal entry DNA, we present a model that explains INO80-specificmonitoring of DNA shape by the A-module, the counter-grip subunit Arp5 and the motor ATPase itself. Consequently, mutual conformational feedback between the submodules yields a specificnucleosome positioning outcome.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Structural Biology, Cryo-EM, Snf2 ATPases, chromatin remodeling, Mot1, Ino80
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 22. Dezember 2022
• 1. Bericht­erstatter:in: Hopfner, Karl-Peter