Periodic Reporting for period 1 - HollidayTrack (Tracking the movement and dynamics of Holliday junctions)
Berichtszeitraum: 2020-04-01 bis 2022-03-31
Our cells are continually challenged by various kinds of DNA damage, which needs to be repaired to avoid loss of genetic information, genomic instability and to prevent cancer development. The most toxic type of DNA damage are DNA double strand breaks (DSBs). Therefore, our cells have developed elaborate DNA repair mechanism to ensure genomic integrity. DSBs can be repaired via two major pathways, non-homologous end joining (NHEJ) or homologous recombination (HR). Whilst NHEJ fuses broken DNA ends and is potentially ‘error prone’, HR is considered to be an ‘error-free’ mechanism of DSB repair. HR occurs between homologous DNA sequences that are provided by the undamaged sister chromatid and can lead to the formation of Holliday junctions (HJs) that covalently link sister chromatids. HJs are mobile and can migrate through homologous sequences, a phenomenon called branch migration. These movements can occur spontaneously or can be enzymatically driven by a DNA translocase or helicase. To date, branch migration has only been studied in vitro due to the lack of tools to detect HJs in cells.
In a cell, HJs must be resolved to allow sister chromatid separation in mitosis. Depending on the processing of the HJ. In humans, two pathways are known to separate the recombining DNA molecules: the first is known as ‘dissolution’ and involves BLM, Topoisomerase III, RMI1 and RMI2 (BTRR). The second pathway is termed ‘resolution’ and involves structure-selective endonucleases that cleave HJs. Two structure selective endonucleases are responsible for HJ resolution in human cells: GEN1 and the SMX complex (SLX1-SLX4-MUS81-EME1-XPF-ERCC1). Defects in dissolution are observed as an increased frequency of sister chromatid exchanges, whereas resolvase-deficient cells in combination with BLM mutations are inviable.
There are several outstanding questions regarding the in vivo properties of HJ intermediates that I intend to answer: 1) How far do HJs branch migrate from the site of DSB formation? 2) What enzymes drive HJ branch migration in vivo? 3) How do HJ processing enzymes influence the HJs branch migration?
In a cell, HJs must be resolved to allow sister chromatid separation in mitosis. Depending on the processing of the HJ. In humans, two pathways are known to separate the recombining DNA molecules: the first is known as ‘dissolution’ and involves BLM, Topoisomerase III, RMI1 and RMI2 (BTRR). The second pathway is termed ‘resolution’ and involves structure-selective endonucleases that cleave HJs. Two structure selective endonucleases are responsible for HJ resolution in human cells: GEN1 and the SMX complex (SLX1-SLX4-MUS81-EME1-XPF-ERCC1). Defects in dissolution are observed as an increased frequency of sister chromatid exchanges, whereas resolvase-deficient cells in combination with BLM mutations are inviable.
There are several outstanding questions regarding the in vivo properties of HJ intermediates that I intend to answer: 1) How far do HJs branch migrate from the site of DSB formation? 2) What enzymes drive HJ branch migration in vivo? 3) How do HJ processing enzymes influence the HJs branch migration?
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.
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.
This project validates for the first time since Robin Holliday’s proposal for the intermediate structure (1964), that HJs exist in human cells and demonstrate their dynamics. This approach addresses several longstanding questions in the somatic DNA recombination field. and provides a tool to study HJs during meiotic recombination and their presence at telomeric ends or after replication fork reversal. Furthermore, it could be used to study HJ formation in different cancer cell types to investigate differences in HJ formation, distribution, and migration length. Once the sequencing of the above-mentioned samples is performed and analysed a manuscript will be published to make the data accessible to the scientific and public field.