WP1) Develop and characterize a molecular tool that specifically binds Holliday junctions in vivo
The main aim was to elucidate the dynamics of HJ migration after DSB formation in human cells. To do this, it is necessary to develop a tool that can be used to recognise, track, and assess HJs within a cellular environment by chromatin immunoprecipitation with deep sequencing (ChIP-seq). We generated HJ specific antibodies by presenting in vitro generated HJs structures to a phage display library. The resulting antibodies from this screen were validated for their specificity and that they are work for the desired application. To test if the antibody can detect HJ in a ChIP experiment an in vitro ChIP assay was developed. Unfortunately, not one of the antibodies had the properties necessary for the HJ ChIP experiment. Therefore, an alternative was developed by using an HJ specific protein from bacteria, which had the necessary specificity for HJs and works in a ChIP experiment.
WP2) Track Holliday junctions following DSB formation in vivo
To assess HJs in a cellular environment, cells stably expressing the HJ binding protein fused to monomeric GFP, to visualise foci, and fused to TurboID to identify the HJ interactome were generated. After DNA damage no foci formation or change in the interactome could be observed, indicating that the HJ is not accessible in a cellular environment, or the formation of HJs is too infrequent to distinguish signal from noise.
WP3) Analyse migration length and the kinetics of Holliday junction movement
To address the questions of HJ migration dynamics, I performed time-dependent chromatin immunoprecipitation with deep sequencing (ChIP-seq) using the Holliday junction specific protein. In order to identify the length and speed of branch migration, it will be important to know the exact location of the initial DSB. Therefore, experiments were performed in synchronized DIvA (DSB inducible via AsiSI) human cells expressing a site-specific endonuclease, AsiSI, which after tamoxifen treatment, localizes rapidly to the nucleus and creates approximately 80 site specific DSBs per human cell with a known origin. After several rounds of protocol optimisation, ChIP enriched HJs could be detected around the DSB sites by sequencing and qPCR. The HJ migration length could be measured for one-time point and compared with other DNA damage repair factors. More time points of S/G2 synchronised as well as G1 or unsynchronised cells have been submitted for sequencing and the analysis is pending.
WP4) Determine the cellular role of Holliday junction processing enzymes
To investigate the contribution of a variety of enzymes that have been shown to influence HJ and their migrate junctions in vitro are deplete via CRISPR-Cas9 mediated knockout in DIvA cells. The ChIP experiments were performed in MUS81 and GEN1 knockouts and submitted for sequencing and the analysis is pending. CRISPR-Cas9 mediated knockout of relevant helicase are ongoing.
