Active Recombinant Human RAD51

• Cat.No.: RAD51-1121H

Specification

• Species: Human
• Source: E.coli
• Tag: Non
• Description: Properly controlled recombination between homologous DNA molecules (Homologous Recombination -HR) is essential for the maintenance of genome stability and for the prevention of tumorigenesis. Rad51 is a mammalian homologue of yeast RAD51 and bacterial RecA and, like its counterparts, plays a central role in HR. Rad51 coats ssDNA to form a nucleoprotein filament that invades and pairs with a homologous region in duplex DNA. It can then activate strand exchange to generate a crossover between the juxtaposed DNA molecules. In addition to Rad51, these steps require the coordinated action of a number of other homologous-recombination proteins, including the RP-A protein, which binds single-stranded DNA, Rad52, which can bind DNA ends, anneal complementary single-stranded DNA molecules and enhance the specificity of RAD51, and a number of Rad51 paralogs. The tumour-suppressor proteins BRCA1 and BRCA2 colocalize with RAD51 in DNA-damage-induced nulear foci. BRCA2 has been shown to interact directly with RAD51 and thus plays a direct role in repair by HR, through control of the availability and function of the central mediator, Rad51. Recombinant RAD51 was expressed in a strain of E.coli and purified by an affinity column in combination with FPLC chromatography.
• Form: Liquid. Supplied in 20 mM Tris-HCl pH 8.0, 100 mM KCl, 0.2 mM EDTA, 1 mM DTT and 20% glycerol.
• Purity: ≥95% by SDS-PAGE.
• Application: RAD51 has been applied in DNA and protein-protein interaction assays.
• Activity: 20 ng are sufficient for a DNA-protein assay and 100 ng are sufficient for a protein-protein interaction assay.
• Usage: For in vitro use only.
• Storage: Quality guaranteed for 12 months store at -80°C. Avoid freeze / thaw cycles.

Gene Information

• Gene Name: RAD51 RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae) [ Homo sapiens ]
• Synonyms: RAD51; RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae); RECA; BRCC5; HRAD51; RAD51A; HsRad51; HsT16930; RAD51 homolog protein; RecA-like protein; RAD51D; recombination protein A; RecA, E. coli, homolog of DNA repair protein RAD51 homolog 1; BRCA1/BRCA2-containing complex, subunit 5; RAD51L3; hRAD51; RAD51 (S. cerevisiae) homolog (E coli RecA homolog)
• Gene ID: 5888
• mRNA Refseq: NM_002875
• Protein Refseq: NP_002866
• MIM: 179617
• UniProt ID: Q06609
• Chromosome Location: 15q15
• Pathway: Homologous recombination; Pancreatic cancer; Pathways in cancer
• Function: ATP binding; damaged DNA binding; double-stranded DNA binding; identical protein binding; nucleoside-triphosphatase activity; nucleotide binding; protein C-terminus binding; single-stranded DNA binding; single-stranded DNA-dependent ATPase activity

Citation

Efficient Zygotic Genome Editing via RAD51-Enhanced Interhomolog Repair

Journal: bioRxiv    Data: 2018/8/6

Authors: Wilde Jonathan J., Aida Tomomi, Feng Guoping

Article Snippet:PrePrint: For experiments using wildtype recombinant proteins, the following proteins were used: RAD51 (Creative BioMart RAD51-134H), USP1/WDR48 (Creative BioMart USP1&WDR48-1067H), BCCIP (Origene TP303061), XRCC1 (TP304952), XRCC4 (Origene TP312684), DMC1 (Origene TP318311).For experiments using wildtype recombinant proteins, the following proteins were used: RAD51 (Creative BioMart RAD51-134H), USP1/WDR48 (Creative BioMart USP1&WDR48-1067H), BCCIP (Origene TP303061), XRCC1 (TP304952), XRCC4 (Origene TP312684), DMC1 (Origene TP318311).. For experiments using mutant RAD51, custom RAD51 preparations were produced by Creative BioMart according to their standard protocol for producing the wildtype RAD51 used in all other experiments.. DNA sequences used for cloning the mutant forms of Rad51 are described in Supplementary Table 2 .DNA sequences used for cloning the mutant forms of Rad51 are described in Supplementary Table 2 .

a, Schematic of strategy for testing RAD51-enhanced IHR. Wildtype C57BL/6N eggs were fertilized in vitro by sperm collected from male Chd2 R1684H/R1684H mice and cultured for 8 hours. At 8hpf, PNI was performed with Cas9 protein, crRNA, and tracrRNA with or without RAD51 protein. Since R1684H mutants carry a mutation in the PAM associated with the crRNA utilized in these experiments, Cas9 is only capable of cutting the maternal allele. Injected embryos were then cultured for 48 hours and collected at morula stage. Half of the purified DNA was used for nested PCR and Sanger sequencing and the other half was used for multiplex PCR and subsequent qPCR to analyze genomic copy number at the Chd2 editing locus. b, Representative chromatograms showing the wildtype reference sequence (Ref., top), an uninjected Chd2 R1684H/+ embryo generated by IVF (Uninj., middle), and a pure Chd2 R1684H/R1684H homozygous mutant (IHR, bottom) generated via donor-free, RAD51-enhanced three-component CRISPR. c, Quantification of Sanger sequencing results from IVF-derived embryos co-injected with or without RAD51. ‘All IHR’ includes mosaic embryos and was determined by identifying embryos with >2:1 ratio of R1684H-to-wildtype alleles (one-tailed chi-square test, p<0.0001). Pure IHR refers to embryos without mosaicism (p=0.018, one-tailed chi-square test). d, Genomic qPCR targeting Shank3B using 180pg genomic DNA from wildtype and Shank3B +/- mice as a control for copy-number sensitivity (lanes 1-2, p=0.00295, unpaired t-test, t=6.47, df=4, n =3 technical replicates per sample, error bars=SEM). Lanes 3-4 illustrate genomic qPCR for the Chd2 R1684H locus using 100% and 50% input of DNA derived from wildtype blastocysts for Chd2 quantification (but 100% input for Gapdh ) as a control for copy-number sensitivity at the locus (p=0.0099, unpaired t-test, t=5.355, df=3.302, n =4 technical replicates per condition, error bars=SEM). Lanes 5-10 illustrate genomic qPCR for the Chd2 R1684H locus using DNA from an uninjected embryo and 5 randomly selected pure homozygous Chd2 R1684H embryos (p>0.05, unpaired t-test, t=4.60, df=6, n=4 technical replicates per sample, error bars=SEM) . e, F 1 genotyping results of litters derived from crosses between wildtype C57BL/6N mice and 5 pure Chd2 R1684H/R1684H F 0 animals generated by IVF with RAD51.