Final Report Summary - INOHR (Chromatin Remodeling in DNA Repair: the role of the INO80 complex) The project aimed to investigate the mechanisms of chromatin remodeling by the human INO80 complex at the single-molecule level. The INO80 complex plays roles in genome integrity maintenance by acting on the homologous recombination pathway of double stranded DNA break repair and responsible for the dynamic distribution of the specific nucleosomes. Beside the scientific aims the Marie Curie Fellowship was intended to significantly develop Dr. Gyimesi’s career by supporting the acquirement of new, regionally unique techniques such as microfluidics coupled single-molecule TIRF microscopy and optical trapping and manipulation of chromatin structures. An important impact of the project was to transfer the acquired knowledge to the Central European region and consolidate the novel techniques at the return Host Institute.Dr. Gyimesi succeeded in expression of six human histones. He cloned nucleosome positioning sequences (NPSs) of three different types and established a cloning strategy to multiply the NPS units to achieve arrays of NPSs of different lengths. He also cloned the recognition site for a specific integrase (PhiC31) into the NPSs containing plasmid constructs. The NPS sequences were integrated into a modified lambda construct (what we named λKytos), which contains the recognition site pair for the integrase. Dr. Gyimesi has also purified the PhiC31 integrase. The integrated lambda DNA constructs with NPS arrays were packaged into phage and the lysogenic strains were developed and maintained. Homogenous and high purity DNA was isolated from the monoclonal phage.Dr. Gyimesi labeled specific human histones with different fluorophores and reconstituted histone octamers from the unlabeled and labeled histone variants. Mononucleosome formation assays were performed with all types of octamers to test the ability of the labeled octamers to form chromatin structure. He also purified fluorescent histone dimer in order to study histone exchange activity of the INO80 chromatin remodeling complex. Dr. Gyimesi carried out optical trap and TIRF microscopy based single-molecule analyses of the chromatin structures formed from the labeled histone octamers. Position analysis of the reconstituted nucleosomes was performed to test the positioning characteristics of the NPS containing lambda substarte. Photobleaching analyses were carried out to gain insight into the number of nucleosomes per fluorescent foci. Dr. Gyimesi attempted to purify both full-length and N-terminally truncated versions of the INO80 complex from HEK293/FRT human cells using FLAG-affinity chromatography. The poor yield and the perturbed ATPase activities of the purified complexes led us to change the expression strategy in a collaborative effort with Prof. Dale Wigely at the Institute of Cancer Research, London, UK. Our results demonstrate that labeling of histones with neither fluorophores affected the ocatmer and nucleosome formation abilities. The NPS arrays could be integrated into lambda DNA with nearly 100% efficiency using the purified integrase. The integrants containing NPS units could be packaged into phage and high purity homogenous DNA could be purified from the lysogenic strains. Nucleosomes could be reconstituted from the labeled octamers, and the biotinylated chromatin structure could be visualized in optical trap and TIRF microscope. Position analysis of the nucleosomes showed that 40% of the nucleosomes bound to the positioning region. Photobleaching analysis showed that the majority of fluorescent spots consisted of 1 or 2 nucleosomes. These results demonstrated that Dr. Gyimesi was able to establish a protocol to routinely prepare substrates for single-molecule studies of chromatin remodeling. Using the these methods for preparing the chromatin substrate suitable for single-molecule studies of chromatin remodeling established by Dr. Gyimesi we now have the possibility to investigate the activities of the INO80 complex. The ongoing efforts in the Kowalczykowski-lab resulted in the visualization of INO80 translocation on DNA and the INO80-dependent movement and eviction of nucleosomes from chromatin structures formed by the method developed by Dr. Gyimesi.Based on these results Dr. Gyimesi has been writing a manuscript with Prof. Kowalczykowski on single-molecule measurements of several λKytos-based DNA constructs and their visualization in TIRF microscopy, which manuscript will be published in the near future.From training purposes Dr. Gyimesi had the opportunity to improve his skills in preparing microfluidic flow-cells for optical trap and TIRF microscopy, performing single-molecule experiments with optical tweezers, TIRF microscopy and atomic force microscopy, reconstituting and manipulating histones and chromatin structures. He also achieved proficiency in lambda phage biology and acquired the know-how of the versatile λKytos system. Dr. Gyimesi also had the opportunity to attend world-class seminars taking place on Davis campus (speakers from UC Berkely, UC San Francisco, Stanford, University of Cambridge, etc.). He also completed thorough courses required to perform safe and appropriate work with lasers, radioactive isotopes, biological samples and medical waste. He attended to the seminar series ‘Advanced Concepts in DNA Metabolism’ held by three famous experts of the DNA recombination field (Prof. Kowalczykowski, Prof. Heyer and Prof. Hunter). As the project enabled Dr. Gyimesi presented his work at the 58th Annual Meeting of the Biophysical Society in San Francisco.During the return phase of the project Dr. Gyimesi set up all protein purification and phage manipulation systems that he acquired at UC Davis. Briefly, he successfully expressed and labeled human histones, and managed to set up the histone octamer reconstitution system, which served as a basis for nucleosome formation. He also purified lambda phage and DNA from λKytos and NPS containing λKytos derivatives and purified the necessary PhiC31 integrase. These systems now enable the Host Department to widen the research fields with single-molecule studies of DNA processing enzymes playing roles in DNA integrity mainenance requiring otherwise inaccessible DNA structures.The return Host Laboratory utilized all the newly obtained skill-sets of Dr. Gyimesi, as new projects regarding DNA repair mechanisms have been initiated by the access of the versatile λKytos-PhiC31 integrase systems. Dr. Gyimesi’s newly acquired skills in single-molecule imaging and TIRF microscopy was exploited in the procurement of a state-of-the-art Nikon TIRF microscope. His know-how in microfluidics-coupled TIRF microscopy and manipulation of lambda DNA and labeling of DNA modifying enzymes will be further used to improve the commercially available microscope body with microfluidic flow-cells and accessory elements required to simultaneously detect three different fluorescently labeled components of complex enzymatic reactions on the single-molecule level.Based on the new skills Dr. Gyimesi managed to get support from the Marie Sklodowska-Curie Actions Reintegration Panel to consolidate his position and the newly acquired techniques at the Host University. Dr. Gyimesi also applied for the ERC Starting Grant with a fully individual project based on his thorough expertise in the enzymology of DNA modifying enzymes and his newly obtained skills in single-molecule imaging of protein-DNA interactions.
