Molekulare und strukturelle Aufklärung der ADP-ribosylierungsabhängigen Aktivierung einer Snf2-ähnlichen Nucleosome Remodeling ATPase

• Applicant: Professor Andreas Gerhard Ladurner, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology
• Term: from 2009 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 158970805

• Final Report Year: 2016

Final Report Abstract

We made good progress in the biochemical and functional characterization of the ATP-dependent and macrodomain containing nucleosome remodeler Alc1/Chd1L. The biochemical data we obtained using in vivo (fluorescence-2-hybrid) and in vitro (ITC, pull down experiments) assays demonstrate an intramolecular interaction between the ATPase domain and the macrodomain of Alc1/Chd1L. This interaction is lost in the presence of poly-ADP-ribose or of tri-ADP-ribose, but not monomeric ADP-ribose. This separates the Alc1/Chd1L macrodomain from other macrodomaincontaining proteins like the human histone variant macroH2A.1.1, which recognizes monomeric, dimeric and trimeric ADP-ribose with the same affinity (our unpublished data). From these results, and since Alc1/Chd1L is also rapidly recruited to DNA damage sites, Alc1/Chd1L could function as a bona-fide sensor for the DNA-damageinduced and PARP1-mediated synthesis of poly-ADP-ribose. We also obtained first data on conformational changes in distinct parts of Alc1/Chd1L upon treatment with tri-ADP-ribose. By applying mass spectrometrycoupled H/D-exchange measurements conformational changes were observed in a region outside the macrodomain and in a region within the macrodomain (our unpublished data). Further analysis will be necessary to reveal the functional consequences of these conformational changes upon tri-ADP-ribose binding. Also the availability of a baculovirus expression facility should enable us to proceed with structural analysis of the Alc1/Chd1L remodeler.

Publications

• Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler. PNAS 2009, 106(33): 13770-13774
  Gottschalk AJ, Timinszky G, Kong SE, Jin J, Cai Y, Swanson SK, Washburn MP, Florens L, Ladurner AG, Conaway JW, Conaway RC
  
• Towards a structural understanding of PARP1 activation and related signalling ADP-ribosyl-transferases. Curr Opin Struct Biol. 2012, 22(6): 721-729
  Hassler M, Ladurner AG
  
• A family of macrodomain proteins reverses cellular mono-ADP- ribosylation. Nat Struct Mol Biol. 2013, 20(4): 508-514
  Jankevicius G, Hassler M, Golia B, Rybin V, Zacharias M, Timinszky G, Ladurner AG
  
• The recognition and removal of cellular poly(ADP-ribose) signals. FEBS J. 2013, 280(15): 3491-3507
  Barkauskaite E, Jankevicius G, Ladurner AG, Ahel I, Timinszky G