Periodic Reporting for period 2 - DNAREPAIRMAN (Regulated Assembly of Molecular Machines for DNA REPAIR: a Molecular Analysis training Network) Reporting period: 2019-01-01 to 2020-12-31 Summary of the context and overall objectives of the project The DNARepairMan training network (Regulated Assembly of Molecular Machines for DNA Repair: a Molecular Analysis Training Network) consists of 12 European participants coming from academia, industry and the creative sector. The network aims to train 12 young scientists in cutting edge biophysical research methodology to address central questions in biology with relevance for human health. This is motivated by the understanding that the future development of novel targeted therapies and antimicrobials will rely precisely on understanding the molecular and mechanistic details underlying disease. Our focus on the process of DNA repair arises from its essential role in maintaining genome stability. In this process multiple proteins collaborate as large molecular machines to detect and repair very rare errors that occur in DNA. These evolutionarily-conserved processes are of fundamental importance, and there is an increasing awareness that targeting of DNA repair pathways could also be useful in the development of personalized anti-cancer therapies and new antimicrobials. This Training Network uniquely brings together biologists, chemists, and physicists around a common research question: What are the mechanistic details that underlie the molecular organization of DNA repair?The objectives of DNARepairMan are- To unravel the mechanistic details of two DNA repair pathways and how they interact with DNA replication and transcription- To develop new reagents, assays, technology and software to analyze fundamental chemical and physical principles- collaborate with an Art Academy to explore new ways and means to visualize research projects and explain the importance of fundamental research to society. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Understanding of the mechanism of communication along DNA: When cells divide, DNA is replicated so that each new cell receives a complete copy of parental genomic information. Copy errors have to be repaired to avoid mutations which might otherwise increase the risk to develop cancer. During this repair process, the protein molecules that carry out the different reaction steps have to communicate between different sites on DNA. The consortium has established that the repair proteins move along the DNA backbone during this search process, and that obstacles on the DNA can be bypassed during this search. This work has been published.The protein that recognizes the replication error, MutS, has to recognize this error, bind to it and only then recruit the next protein in the repair cascade. During this process MutS changes shape multiple times. Using a high resolution technique called cryo-electron microscopy, the consortium has taken snapshots of these different states, which give unprecedented detail and explain how MutS does its important job.Understanding which proteins collaborate at which reactions steps and how they do this: During the DNA repair process, many proteins collaborate to 1) find copy errors or damage in the DNA, 2) recruit partner proteins that can remove the error or damage, and 3) recruit proteins that can restore the original information. In order to understand the molecular mechanism of these processes, the consortium has designed new reagents, assays and technologies. These are used to study the mechanism of the repair processes in detail.The training program: The training program is an essential and central component of DNARepairMan. The consortium has organized four scientific workshops, which have been attended by all the fellows as well as several researchers from outside the consortium. The workshops entailed ‘Protein Structure Analysis’, ‘Advanced single molecule analysis’, ‘Fluorescence and cross linking approaches’ and 'Hybrid structural biology. Fellows have also received entrepreneurship coaching sessions to prepare them for a putative career as entrepreneur, a lecture on Research Ethics, as well as other transferable skills and a career event.DNARepairMan is a truly interactive consortium; all the young researchers have already spend prolonged research visits (secondments) in each other’s laboratories to learn new techniques and to collect data.A unique component of DNARepairMan is the active collaboration with the Art Academy in Rotterdam (de Willem de Kooning Akademy). Through an intensive program including the DNArepairMan fellows, scientists, students and teachers from the Art Academy and the Erasmus MC, the consortium is exploring new ways and means to communicate scientific topics and results as well as the importance of fundamental science in general to society. This has resulted in several short animations about the scientific projects and the importance of science in general. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The consortium has designed, created and benchmarked multiple reagents, assay and technologies that go beyond the state-of-the-art and have used those to answer questions regarding the molecular mechanism of multiple DNA repair pathways that are relevant for human health:• DNA molecules carrying specific errors or DNA damage combined with fluorescent labels that allow readout of mechanisms of communication and achieving directional DNA repair• Purified protein molecules that carry fluorescent labels that allow quantitative analysis with interaction partners.• Purified protein molecules with functional groups that allow cross linking interaction analysis.• Purified protein variants that carry changes in structure to analyze functional importance• Single molecule assays that allow detailed identification of binding partners and kinetics of reaction steps• Microfluidic flow cells that allow controlled reagent addition during single molecule analysis• New fluorescent assays to quantify protein interactions• New biochemical assays to quantify DNA repair with purified components.The abovementioned investments in beyond-the-state-of-the-art developments and extensive training of the young DNARepairMan researchers have lead to 1) important new understanding of the molecular mechanism underlying DNA repair with impact on human health, and 2) a new generation of innovative young scientists which has been trained in cutting-edge biophysics and with affinity for the academic, industrial and creative sector. 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