1-1 Homologous recombination (HR)
Homologous recombination (HR) is an important process for maintaining the gene integrity and producing the gene diversity. DNA in cell undergoes double strand break (DSB) when exposed to UV irradiation, chemical environment, or in the sister chromatid pairing in cell division1.
The DSB phenomena could lead to apoptosis without repairing. In HR, the DSB DNA will be degraded to generate a 3' single strand overhang by nuclease and helicase.
Then, the recombinases facilitate the homologous DNA sequence searching by forming presynaptic filament with the 3' overhang DNA. The presynaptic filament formation process can be divided into three steps. First, recombinases form a stable nucleus on ssDNA. Second, free recombinases assemble on the ssDNA from the nucleus and extend the filament. Finally, the filament covers most of the ssDNA and continue the homologous sequence searching. The first part, nucleus formation, is the rate determining step2. The synaptic filament surveys the homologous sequence and invades in it to become a displacement loop (D-loop) for the following strand exchange process. After searching the homologous DNA sequence, the injured DNA use the intact homologous DNA as a template to undergo the replication for the repairing purpose.
Figure 1-1 Double-strand break repair by the homologous recombination The DSB will first be degraded as 3' overhang. With the recombinases and accessory protein help, the injured DNA can search for the homologous sequence for replication and repair. The proposed products of the repaired DNA have two type: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR)3.
1-2 Dmc1 and Rad51 Recombinase
In prokaryote cell, only RecA plays a role of recombinase in HR. On the contrary, in eukaryote cell, there are two recombinases, Rad51 and Dmc1, processing in HR. Patrick Sung and Eric Greene have compared many types of the recombinases in the kinetic process of homologous DNA searching. For E.coli RecA, ScDmc1, and ScRad51, the smallest presynaptic filament searching the homologous sequence in HR process is about 8 nts in size, and all of them share the same stoichiometry ratio of 3 nucleotides to each recombinase4. For the homologous recombinases Rad51 and Dmc1, although the amino acid sequence is highly conserved3, 5, many biochemical assays show their difference.
In cell cycle, Rad51 can function in both meiotic and mitotic cell, while Dmc1 is meiotic-specific. The mechanism underlying these different requirements is still unknown.
During the homologous sequence searching in HR, Dmc1 has higher endurance in mismatch sequence pairing than Rad51 and RecA6, 7. Some previous studies indicate the different DNA-protein structures or functions between these two recombinases. In EM image, both ScDmc1-DNA and hDmc1-DNA interaction have two kinds of filament formation. One is the general extended filament structure as the RecA filament, and the other is a stack-ring structure5, 8, 9. However, the stack-ring structure is absent in Rad51-DNA interaction.
Based on those observations, Rad51 is considered to dominate the general repair process in HR because of the high fidelity of DNA sequence searching. However, owing
single molecule level.
Figure 1-2 Similarities and differences among recombinases from different species.
(A) Measuring the dissociation rate with a fluorophore labeled presynaptic filaments which have different homology lengths DNA, the binding signal starts to appear when the homology presynaptic filament increases to 8 nts10. (B) The filament binding energy change every 3 nucleotides, hint each recombinase bind 3 nucleotides7.(C) With a single mismatch nucleotide, only Dmc1 can endure the mismatch in filament pairing7. (D) Top:
Electron microscopic visualization of hDmc1-ssDNA shows the filaments conformation with ATP. Bottom: Electron microscopic visualization of hDmc1-ssDNA shows the stack-ring conformation with ATP. The magnification bars represent 50 nm11.
1-3 Calcium modulates the activity of Rad51 and Dmc1
The calcium concentration is a signal for cell cycle. Especially in meiosis, the surge of calcium concentration is essential12. In the coincidence of Dmc1 existence and the increased calcium concentration, calcium might affect the activity of Dmc1. Both hDmc1, hRad51, and ScDmc1 D-loop formation and strand exchange can be improved by Ca2+
appearance13, 14, 15. Although the ATP-hydrolyzing activity of both hDmc1 and hRad51 are suppressed, the fact that the activation of D-loop assay has a higher dependence on free Ca2+ than Ca-ATP for hDmc1 indicates the possibility of the second Ca2+ binding site on Dmc114.
Figure 1-3 Calcium ions affect the hRad51 and hDmc1 joint molecule formation.
(A) For hRad51, the Calcium ion can improve the formation of the joint molecule. While the enhancement doesn’t appear on yRad5113.
(B) Top: At a certain ca concentration, the hDmc1 joint molecule has the highest signal of the product. Bottom: Although the Ca-ATP can suppress the ATP hydrolysis, there is no obvious tendency between the joint molecules formation and Ca-ATP concentration.
The above results indicate that Dmc1 might have the second ca binding site14.
1-4 Motivation of this study
The meiosis process needs the existing of both Rad51 and Dmc1. But the reason why the two recombinases need to coexist in the meiosis is still unknown.
Although many studies have worked on the overall HR reaction to find the difference between Dmc1 and Rad51, the important and essential step, presynaptic filament formation is rarely investigated. To illustrate the roles in the meiosis of the two homologous proteins, comparing the kinetic properties in the presynaptic filament formation which is the essential step in HR is a good starting spot.
The unpublished data from our collaboration lab indicates the ScRad51-ssDNA filament is more stable than ScDmc1’s. Here, we aim to compare the different kinetic properties between these two recombinases in filament formation by single molecule method- tethered particle motion (smTPM). Besides, we would like to confirm the interaction among ScRad51 and ScDmc1 whether can improve the presynaptic filament formation or not.