Periodic Reporting for period 1 - HJMIGRA (Single-molecule analysis of Holliday-junction (HJ) migration by the human double-HJ dissolvasome)
Okres sprawozdawczy: 2015-05-01 do 2017-04-30
The project objective was to investigate the molecular mechanisms of the critical final steps of homologous recombination (HR) based DNA repair. HR enables the cells to maintain genome integrity as a prerequisite for avoiding cancerous transformations.
One important pathway of HR is the double-stranded DNA break repair (DSBR) that employs an enzyme complex (‘dissolvasome’) to dissolve the formed double Holliday junction (DHJ) intermediate structure in a solely non-crossover manner, which thereby helps to maintain chromosome integrity. Although the key proteins in the dissolution reaction have been described, the precise molecular mechanism of HJ migration has not been clarified in details, yet. One major aim of the project aimed to design and purify a mobile HJ substrate and follow dissolvasome mediated branch migration at the single molecule level. Thus, we setup a multichannel detection total internal reflection fluorescence (TIRF) microscope combined with microfluidic flow-cells. This helps us to elucidate the molecular events during HJ migration and the roles of the complex components in this important biological process.
One important pathway of HR is the double-stranded DNA break repair (DSBR) that employs an enzyme complex (‘dissolvasome’) to dissolve the formed double Holliday junction (DHJ) intermediate structure in a solely non-crossover manner, which thereby helps to maintain chromosome integrity. Although the key proteins in the dissolution reaction have been described, the precise molecular mechanism of HJ migration has not been clarified in details, yet. One major aim of the project aimed to design and purify a mobile HJ substrate and follow dissolvasome mediated branch migration at the single molecule level. Thus, we setup a multichannel detection total internal reflection fluorescence (TIRF) microscope combined with microfluidic flow-cells. This helps us to elucidate the molecular events during HJ migration and the roles of the complex components in this important biological process.
The project consisted of four Work packages (WPs): WP1 – Protein biochemistry, WP2 – λHJ substrate preparation, WP3 – Instrument building, WP4 – Single molecule visualization of HJ structure and HJ migration. The project was successful in all four WPs, however, further measurements are required to characterize the HJ migration reaction.
WP1 – Protein biochemistry. Bloom’s syndrome DNA helicase (BLM), its monomeric form and the bacterial BLM homolog (RecQ) were expressed and purified. I developed methods to fluorescently label all three purified helicases at their N-terminus.
I expressed and purified TOP3A, RMI1 and RMI2 as a complex (TRR). I also purified the catalytically dead TOP3A containing TRR complex and the heterotrimeric human ssDNA binding protein (RPA). ATPase measurements suggested that BLM in complex with TRR and RPA (dissolvasome) could partially melt λDNA, which was important if we planned to visualize BTRR+RPA complex movements along λDNA-based HJ substrate.
I also expressed and purified active φC31 integrase that was to integrate a HJ containing plasmid into the middle of λDNA in my original proposal (see WP2 below).
WP2 – λHJ substrate preparation. The original concept to generate a single molecule visualization-ready substrate containing a HJ structure was designed with the use of a λDNA construct containing a recognition sequence for φC31 integrase in the middle of λDNA (λKytos). However, several rounds of annealing and ligation resulted in undetectable amount of HJ containing plasmid that could have been integrated into λKytos.
The new strategy was based on dimer formation from a digested plasmid. Digested plasmid could be self-ligated to generate a head-to-head ligated doublet. The doublet thus formed a fully mobile HJ structure on ~19 kbp length. The doublet and λKytos was digested with EcoRI and ligated to form the λHJ structure.
PCR assay that the ratio of λHJ structure in the ligation product was cca. 35%. This result was reproducible and demonstrated that the new ligation based method could be used to produce the λHJ structure that could be visualized in the TIRF microscope.
WP3 – Instrument building and improvement. During the project period I was in charge with the improvements of the a 543-nm laser line containing Nikon Eclipse Ti-E TIRF microscope with two more laser lines (488-nm and 642-nm) and a microfluidic flow system with the support of the project Supervisor’s (Mihály Kovács) other grants. I succeeded to obtain the laser lines that now enable us to parallely follow three differently labeled objects (e.g. DNA and two protein components) in the TIRF microscope.
I also managed to design, build and assemble the valve system responsible for loading and manipulating solutions in the microfluidic flow cells.
I also managed to fully adapt the DNA immobilization and imaging techniques I acquired during my previous Marie Curie Fellowship at UC Davis.
WP4 – Single molecule visualization of HJ structure and HJ migration. A striking phenomenon was observed when λHJ was visualized under the same conditions as λKytos. While the DNA molecules in the λKytos sample were invariant in length and shape, DNA molecules in the λHJ sample contained an intensively stained spot that was never observed on λKytos molecules. The presence of the spots was indicative that some kind of structure has been formed in the molecules that were never seen in simple λKytos samples. Analysis of the position of the spots showed that the spot is 7 μm from the end of the molecules. This is half-length of λDNA where the EcoRI site takes place, i.e. the place where pEG doublet was ligated, in other words where we expect the HJ structure to be formed. These results indicated that a new structure was formed in a position where the HJ structure was ligated.
Other training objectives – I was an invited speaker at the Mechanism of Genome Maintenance international symposium in Davis, California. My talk covered our work with the BLM helicase, the single molecule studies I’ve carried out during my former Marie Curie Fellowship and the current HJ migration project. I also attended at the Annual Meeting of the Biophysical Society where I presented the results of the project including HJ structure design and preparation and the single molecule analysis of the HJ structure.
I conducted workshops for students and postdocs on single molecule imaging with TIRF microscope. During these workshops people brought their own samples and, at the end of the practical part, they managed to use the microscope independently.
I was a lecturer of the “Genetechnology and Protein Engineering” course in the Master program of our University where single molecule techniques with special interest in single molecule visualization have been demonstrated.
Single molecule visual biochemistry techniques was the topic of my “Budapest Science Meetup” talk at the Hungarian Academy of Sciences. I also went to advertise science and the scientist lifestyle to high school students as part of their granting program.
During the project period we managed to publish two scientific papers in Methods and PNAS. The Methods paper summarized the fluorescent methods that we use to study the mechanochemistry of DNA helicases and translocases. The PNAS paper described the shuttling mechanism and the directed processing of HR intermediate DNA structures, which processes are necessary to perform quality control activities of RecQ helicases during DNA repair functions. These works provide a well-defined basis for the elucidation of HJ migration results coming out from the single molecule studies in the future.
WP1 – Protein biochemistry. Bloom’s syndrome DNA helicase (BLM), its monomeric form and the bacterial BLM homolog (RecQ) were expressed and purified. I developed methods to fluorescently label all three purified helicases at their N-terminus.
I expressed and purified TOP3A, RMI1 and RMI2 as a complex (TRR). I also purified the catalytically dead TOP3A containing TRR complex and the heterotrimeric human ssDNA binding protein (RPA). ATPase measurements suggested that BLM in complex with TRR and RPA (dissolvasome) could partially melt λDNA, which was important if we planned to visualize BTRR+RPA complex movements along λDNA-based HJ substrate.
I also expressed and purified active φC31 integrase that was to integrate a HJ containing plasmid into the middle of λDNA in my original proposal (see WP2 below).
WP2 – λHJ substrate preparation. The original concept to generate a single molecule visualization-ready substrate containing a HJ structure was designed with the use of a λDNA construct containing a recognition sequence for φC31 integrase in the middle of λDNA (λKytos). However, several rounds of annealing and ligation resulted in undetectable amount of HJ containing plasmid that could have been integrated into λKytos.
The new strategy was based on dimer formation from a digested plasmid. Digested plasmid could be self-ligated to generate a head-to-head ligated doublet. The doublet thus formed a fully mobile HJ structure on ~19 kbp length. The doublet and λKytos was digested with EcoRI and ligated to form the λHJ structure.
PCR assay that the ratio of λHJ structure in the ligation product was cca. 35%. This result was reproducible and demonstrated that the new ligation based method could be used to produce the λHJ structure that could be visualized in the TIRF microscope.
WP3 – Instrument building and improvement. During the project period I was in charge with the improvements of the a 543-nm laser line containing Nikon Eclipse Ti-E TIRF microscope with two more laser lines (488-nm and 642-nm) and a microfluidic flow system with the support of the project Supervisor’s (Mihály Kovács) other grants. I succeeded to obtain the laser lines that now enable us to parallely follow three differently labeled objects (e.g. DNA and two protein components) in the TIRF microscope.
I also managed to design, build and assemble the valve system responsible for loading and manipulating solutions in the microfluidic flow cells.
I also managed to fully adapt the DNA immobilization and imaging techniques I acquired during my previous Marie Curie Fellowship at UC Davis.
WP4 – Single molecule visualization of HJ structure and HJ migration. A striking phenomenon was observed when λHJ was visualized under the same conditions as λKytos. While the DNA molecules in the λKytos sample were invariant in length and shape, DNA molecules in the λHJ sample contained an intensively stained spot that was never observed on λKytos molecules. The presence of the spots was indicative that some kind of structure has been formed in the molecules that were never seen in simple λKytos samples. Analysis of the position of the spots showed that the spot is 7 μm from the end of the molecules. This is half-length of λDNA where the EcoRI site takes place, i.e. the place where pEG doublet was ligated, in other words where we expect the HJ structure to be formed. These results indicated that a new structure was formed in a position where the HJ structure was ligated.
Other training objectives – I was an invited speaker at the Mechanism of Genome Maintenance international symposium in Davis, California. My talk covered our work with the BLM helicase, the single molecule studies I’ve carried out during my former Marie Curie Fellowship and the current HJ migration project. I also attended at the Annual Meeting of the Biophysical Society where I presented the results of the project including HJ structure design and preparation and the single molecule analysis of the HJ structure.
I conducted workshops for students and postdocs on single molecule imaging with TIRF microscope. During these workshops people brought their own samples and, at the end of the practical part, they managed to use the microscope independently.
I was a lecturer of the “Genetechnology and Protein Engineering” course in the Master program of our University where single molecule techniques with special interest in single molecule visualization have been demonstrated.
Single molecule visual biochemistry techniques was the topic of my “Budapest Science Meetup” talk at the Hungarian Academy of Sciences. I also went to advertise science and the scientist lifestyle to high school students as part of their granting program.
During the project period we managed to publish two scientific papers in Methods and PNAS. The Methods paper summarized the fluorescent methods that we use to study the mechanochemistry of DNA helicases and translocases. The PNAS paper described the shuttling mechanism and the directed processing of HR intermediate DNA structures, which processes are necessary to perform quality control activities of RecQ helicases during DNA repair functions. These works provide a well-defined basis for the elucidation of HJ migration results coming out from the single molecule studies in the future.
Genetic instability contributes to cancer and other diseases. Now that we have the reproducible protocol to generate the HJ structure the Kovacs lab has the opportunity to further elucidate HJ migration that will shed light on molecular events supporting genome stability. The results may also provide new opportunities in the development of new cancer treatment methods.
Beside the aforementioned socio-economic impact the project had an exceptional effect on my personal career as I was offered to be a leading scientist in Hungary’s most promising scientific consortium investigating enzyme inhibitors in neuronal plasticity.
Beside the aforementioned socio-economic impact the project had an exceptional effect on my personal career as I was offered to be a leading scientist in Hungary’s most promising scientific consortium investigating enzyme inhibitors in neuronal plasticity.