RecA

• Promotes strand exchange of single-stranded DNA with homologous duplex DNA
• A RecA–ATP single-stranded DNA complex can function as a co-protease
• Complexed with site-specific oliognucleotides can be used to target and specifically cleave large DNA fragments

RecA, which functions in DNA recombination and DNA repair (1, 2, 3), binds to single stranded DNA, resulting in the polymerization of RecA into a nucleoprotein complex. This complex aligns with complementary-double stranded DNA, resulting in RecA catalysis of DNA strand exchange. RecA DNA binding is stimulated by ATP hydrolysis or non-hydrolyzable ATP analogs. The RecA–ATP–single-stranded DNA complex also functions as a coprotease factor in the proteolytic cleavage of LexA, UmuD and certain bacteriophage proteins. RecA complexed with site-specific oligonucleotides has been used to target and specifically cleave large DNA fragments (4).

This enzme is supplied in 10 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C.

10X RecA Reaction Buffer (cat. no. B9260) contains 700 mM Tris-HCl, 100 mM MgCl₂ and 50 mM DTT; pH 7.6 at 25°C.

SDS available upon request.

Enzyme properties

• Storage temperature: –25°C to –15°C
• Molecular weight: 37,973 Daltons

Source of recombinant enzyme protein
The protein is produced by an E. coli strain overexpressing the E. coli recA gene from a plasmid.

Unit definition: Sold by mass of pure protein determined at OD₂₈₀ (A₂₈₀ = 0.516 at 1 mg/m, 1cm).

References

1. Cox, M.M. (2007) Crit. Rev. Biochem. Mol. Biol. 42:41.
2. Bell, C.E. (2005) Mol. Microbiol. 58:358.
3. Xing, X., Bell, C.E. (2004) J. Mol. Biol. 342:1471.
4. Ferriny, LJ., Camerini-Otero, R.D. (1991) Science 254:1494.

Quality control analysis

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Non-specific RNAse contamination is assessed using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Mutagenesis
• Affinity capture of cDNA