4-1 Conclusion
ScRad51 and ScDmc1 have different kinetic properties on filament formation and different preference of DNA substrates.
With smTPM, we analyze the presynaptic filament formation on ssDNA with the recombinases. The kinetic process of ScRad51 presynaptic filament is much faster than the one of ScDmc1. We have known that ScRad51 is responsible for the DNA damage repair, while Dmc1 mainly focus on creating gene diversity in normal cells. Considering the DSB in mitotic cells are urgent to be renovated, Rad51 must have high fidelity and efficiency to repair the injured DNA. The difference in the nucleation rate between ScRad51 and ScDmc1 is reasonable. However, because the meiosis cell is aimed at producing the gene diversity for evolution, maintaining the gene integrity is not the first priority for Dmc1. Therefore, Dmc1 does not need to proceed the HR as fast and efficient as Rad51. This might explain their difference of nucleation rate and DNA binding affinity.
By matching the recombination titration and the nucleation rate, the reaction order of the nucleation reaction or the nucleus size is determined. Interestingly, both ScRad51 and ScDmc1 share similar nucleus size between mono-cluster and di-cluster.
ScRad51 and ScDmc1 have different preference nucleation sites
The different nucleation rate mainly results from ScRad51’s higher affinity of ssDNA than ScDmc1. However, ScDmc1 shows special affinity on the ds/ss junction.
The junction preference of ScDmc1 have been verified with two sets of experiment. With 1. DNA substrates with different lengths of ssDNA and the same number of DNA gap and 2. DNA substrates with the same DNA lengths but different number of DNA gap, we
confirm the special affinity of ScDmc1 and DNA junctions. The dramatic kinetic difference in nucleoprotein filament assembly might be responsible for the requirement of both Rad51 and Dmc1 in meiotic homologous recombination seen in vivo.
ScDmc1-DNA interaction has different modes comparing with ScRad51-DNA.
In single molecular scale, we find that ScDmc1 has the unique behaviors, condensation, and resurgence, in contrast to the typical filament formation of ScRad51.
Because that the Brownian Motion decrease of ScDmc1 can be differentiated from the stuck bead events and that the phenomena are independent of the surface, the BM decrease indicates the special interaction between ScDmc1 and DNA. Considering that the Dmc1 stack-ring structure observed under the electronic microscope, this condensation in smTPM could be related to the special structure in EM. Besides, because the BM surge after the resurgence is similar to the typical presynaptic filament, the condensation could be an intermediate state of the filament formation.
Ca2+facilitate the assembly of ScDmc1 nucleus.
In the meiotic cell, it has been shown that the concentration of calcium ion would increase when meiotic cells become matured. Furthermore, calcium ion is also an indicator that if a huge amount of DNA double-strand break occur inside the cell, the concentration of calcium ion would increase. Based on previous knowledge, calcium ion should be a regulator of homologous recombination inside the cell. It has been supposed
with ATP. Although the result can’t confirm the second binding site for Ca2+ in ScDmc1, this observation could ensure that modulation from Ca2+ promotes the assembly step of ScDmc1 presynaptic filament formation.
The interaction between Rad51 and Dmc1 doesn’t affect the kinetic process of nucleation.
Although previous works have found Rad51 and Dmc1 have interaction both in vivo10 and in vitro27, 28, we didn’t see significant acceleration on Dmc1’s filament formation with Rad51 even at 30oC, which is the temperature that ScDmc1 has physical interaction with ScRad51(data not shown). One possible reason is that the interaction between these two similar proteins doesn’t work on the filament formation. The other is that Rad51 can be an accessory protein of Dmc1 only when the recombinase activity of Rad51 is suppressed, considering that the enhancement from Rad51 is observed only when the suppressor protein Hed1 or the mutant type Rad51 show up27, 28.
Figure 4-1 The hypothesis scheme represents the recombinase presynaptic filament formation.
When a lot of 3' overhang DNA are exposed in meiosis cells, both ScRad51 and ScDmc1 form stable nuclei on ssDNA in dimer. However, ScRad51 prefers to nucleate on the ssDNA part, while Dmc1 prefer to nucleate on the ds/ss junction. In contrast to that ScRad51 directly construct the presynaptic filaments, Dmc1 and DNA were observed to have a meta-stabled state, the condense-ring, before the presynaptic filament formation.
Ca2+ can accelerate the nucleation step of Dmc1.
4-2 Outlook
Dmc1 and Rad51 are important in the mitotic and meiotic process in Eukaryotic cells.
However, the biological meaning of these two similar protein existence still waited to be investigated. Here, we compare ScRad51 and ScDmc1’s kinetic process in presynaptic
overhang DNA with FRET pair on the ssDNA. When the filament formation on DNA overhang, FRET efficiency will decrease owing to the distance between dye pair increasing from the filament DNA stretching. Titrating the length of ssDNA overhang, when the length of the ssDNA is longer than the smallest nucleation size of recombinases, the probability of FRET decrease will incline. With this experiment, we can make sure the nucleation size of recombinases.
Although in kinetics analysis ScRad51 doesn’t enhance ScDmc1 filament formation, the situation might result from that the interaction might not directly affect the filament formation process. To evaluate the viewpoint, both optical tweezer and fluorescent microscopy are powerful tools. With optical tweezer, the real-time force change along the DNA structure can be attained29. Owing to the persistence length and contour length of DNA can reflect the structure of DNA, the optical tweezer might examine the difference between Rad51 filament and Dmc1 filament or the existence of Rad51-Dmc1 co-filament.
The optical tweezer also can measure the stability of filament with offering different force to the filament. Concerning the physical interaction among proteins might not affect the result of functional assays, the fluorescent microscope is the proper tool to see the protein physical interaction by detecting the location of fluorophore-labeled protein. Observing the co-localization of the fluorescent dye from different kinds of protein and the existing time spot of the dye, the protein interaction can be studied. With the fluorescent microscope, the interaction of ScRad51, ScDmc1 and ScMei5-Sae3 can be investigated.
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