Final Report Summary - ATMMACHINE (Structural mechanism of recognition, signaling and resection of DNA double-strand breaks)
The central goal of this project was to use integrative structural biology along with other approaches to reveal the structures and mechanisms of key macromolecular complexes involved in the sensing, signalling and processing of DNA double-strand breaks. DNA double-strand breaks are among the most severe types of DNA lesions and need sensitive detection and highly efficient repair by one of several pathways in order to avoid cancerogenic chromosomal aberrations or cell death. Sensing and signalling of DNA double-strand breaks by the Mre11-Rad50-Nbs1 ATM/ATR axis elicits a very complex cellular response that includes processing and resection of DNA ends, alteration of chromatin structure at DNA ends and nearby chromatin and a carefully orchestrated choice of DNA repair pathways through the regulated formation of distinct chromatin and DNA associated macromolecular assemblies. Underlying these highly regulated but extremely complex macromolecular reactions are large, transient and highly dynamic protein complexes that were poorly understood at the structural and mechanistic level. To reveal the structural and molecular mechanisms and to unravel the molecular choreography underlying DNA double-strand break repair, a critical and ambitious part of the project was the implementation and utilisation of hybrid structural biology methods. Here, we particularly combined X-ray crystallography, electron microscopy, small angle X-ray scattering and chemical crosslinking and mass spectrometry, which allowed us to reveal the dynamic assembly and molecular mechanism of several key DNA repair and chromatin complexes. We determined mechanisms of the Mre11-Rad50 complex in detecting and processing DNA ends. The structures uncovered interesting and unexpected features such as a lack of direct DNA end binding by Rad50, suggesting that Mre11 is the DNA end recognition subunit of the complex. Moreover, our structural analyses revealed how ATP and DNA binding to Rad50 are structurally and functionally coupled and allowed formulation of a unified allosteric model for ABC ATPases. We furthermore determined the architecture of large multisubunits macromolecular complexes involved in chromatin alterations at DNA breaks and DNA end resection, in particular pioneering structures of HerA-NurA resectosome as well as pioneering first insights into the architecture and dynamics of the INO80 chromatin remodeller that plays key roles in DNA double-strand break repair. Latter was the first near-atomic resolution structure of any multisubunit remodeller bound to a nucleosome and clearly a breakthrough in the genome maintenance and chromatin fields. Important results are a multivalent nucleosome recognition mode and insights how the ATP dependent motor domain pumps DNA into the nucleosome to catalyse sliding and editing reactions. In addition, we could clarify the role of actin related proteins in DNA sensing and show that they form a molecular ruler that helps place nucleosomes at appropriate distances from DNA ends during break repair as well as in spacing other nucleosomes. We could also help clarify also a role INO80 in genome maintenance through characterising its ole of ATR in replication-transcription conflicts. An new discovery was a cytosolic role of the Rad50 DNA break sensor in innate immune sensing of DNA, which led to the identification of an unexpected mechanistic link between the DNA repair and innate immune signaling machinery that could have a quite important potential in the understanding and therapeutic exploitation of innate immune signals in cancer development and therapy.