Kinase experiment:

Briefly, 15 μL reaction volumes include a DNA strand exchange protein (0.8 μM) that is preincubated for 5 min at 37℃ with 1 μM (nucleotide concentration) 32P-labeled oligonucleotide 306.7 in a reaction buffer containing 20 mM Hepes (pH 7.5), 1 mM DTT, 2 mM nucleotide cofactor, and 1 mM MgCl2 and various concentrations of RS-1. For experimental buffer conditions that included calcium, 1 mM CaCl2 is present in addition to (in the case of hRAD51) or in the place of (in the case of RecA and scRAD51) the 1 mM MgCl2. Conditions with scRAD51 additionally contains 110 nM scRAD54. After this initial binding reaction, 10 μL of 19.75 μM (base pair concentration) supercoiled homologuecontaining target plasmid DNA (pRS306) is next added along with sufficient magnesium acetate to give a final concentration of 10 mM[1].

RS-1 is a stimulatory compound of RAD51, a key player in the HR complex. RS-1 could increase the DNA binding activity of RAD51 and function in vivo to enhance the homologous recombination activity of hRAD51 by promoting the formation of active presynaptic filaments [1].

The hRAD51 protein could bind with single- and double-stranded DNA and exhibit DNA-dependent ATPase activity. Rad51 mediates homology recognition and initiates strand exchange [2]. Chromosome analysis revealed that Rad51‐deficient vertebrate cells accumulated chromosomal breaks in the G2/M phase of the cell cycle prior to cell death. Rad51 played an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes [3].

RS-1 treatment promoted significant antitumor responses in a mouse mode. It can be used to kill human cancer cells, and that its toxicity is modulated by both RAD51 and RAD54 translocase expression levels [4]. RS-1 effectively improved the knock-in rates in the TALEN- and the Cas9-mediated genome-editing systems in rabbits. RS-1 showed little toxic effects on the overall animal health and reproduction. RS-1 enhanced Cas9- and TALEN-mediated knock-in efficiency in rabbit embryos both in vitro and in vivo. RS-1 treatment (15 μM) appeared to enhance the blastocyst development in embryos. Treating embryos with RS-1 at 7.5 μM resulted in 26.1% knock-in rate [5].

References:
[1]. Jayathilaka K, Sheridan S D, Bold T D, et al. A chemical compound that stimulates the human homologous recombination protein RAD51[J]. Proceedings of the National Academy of Sciences, 2008, 105(41): 15848-15853.
[2]. Baumann P, Benson F E, West S C. Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro[J]. Cell, 1996, 87(4): 757-766.
[3]. Sonoda E, Sasaki M S, Buerstedde J M, et al. Rad51‐deficient vertebrate cells accumulate chromosomal breaks prior to cell death[J]. The EMBO journal, 1998, 17(2): 598-608.
[4]. Mason J M, Logan H L, Budke B, et al. The RAD51-stimulatory compound RS-1 can exploit the RAD51 overexpression that exists in cancer cells and tumors[J]. Cancer research, 2014, 74(13): 3546-3555.
[5]. Song J, Yang D, Xu J, et al. RS-1 enhances CRISPR/Cas9-and TALEN-mediated knock-in efficiency[J]. Nature communications, 2016, 7.