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Single-molecule analysis of Holliday-junction (HJ) migration by the human double-HJ dissolvasome

Periodic Reporting for period 1 - HJMIGRA (Single-molecule analysis of Holliday-junction (HJ) migration by the human double-HJ dissolvasome)

Reporting period: 2015-05-01 to 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.
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 TRR 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
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.
Design and visualization of the λHJ structure