RecA-AC: single-site cleavage of plasmids and chromosomes at any predetermined restriction site

Abstract

We have developed a novel version of the Achilles' Cleavage (AC) reaction in which virtually any restriction site on DNA of any size can be converted to a unique cleavage site. We first polymerized RecA protein on a synthetic oligodeoxyribonucleotide (oligo) in the presence of a nonhydrolyzable ATP analogue to generate oligo:RecA nucleoprotein filaments. These filament were then incubated with plasmid or intact chromosomal DNA from Saccharomyces cerevisiae to form stable complexes in the yeast LEU2 gene at the target sequence identical (or complementary) to that of the oligo. When HhaII (HinfI) methyltransferase (M.HhaII) was added, all of the recognition sites for HhaII with the exception of the one protected by the RecA filament were methylated and thus no longer cleaved by the cognate restriction endonuclease (HinfI). After inactivation of the RecA and the M.HhaII, HinfI was used to efficiently cleave the plasmid or chromosome specifically at the targeted restriction site. Since oligos specific for any sequence can be easily synthesized and the other reagents necessary to perform RecA-mediated AC (RecA-AC) reactions on both plasmids and intact chromosomes are readily available, this procedure can be applied immediately to the precise dissection and analysis of genomic DNA from any source and to any other research problem requiring efficient, highly specific cleavage of DNA at predetermined sites.

Physical mapping of bacterial genomes

“…The actual integration of such a sequence is not necessary because DNA stretches capable of forming such structures can be found in the sequences of most target organisms. By adding RecA to a similar system, one can use an oligonucleotide complementary to any target DNA (45).…”

Expression Analysis of Cloned Chromosomal Segments of Escherichia coli

“…For the remainder, cloning is a more difficult task and it will be necessary to modify our protocol. We will attempt the remainder with the Achilles cleavage technique (8), in which the internal EcoRI sites are preferentially methylated to block cutting prior to digestion of the flanking EcoRI sites and subcloning. It will be necessary to use this and other techniques as well as other sources of inserts to finish cloning the chromosome for expression.…”

Murine MHC class II locus control region drives expression of human β-glucocerebrosidase in antigen presenting cells of transgenic mice

“…The gene for human ␤-glucocerebrosidase was isolated from plasmid PCN 9RI (a generous gift from E Ginns, Bethesda, MD, USA) as a 9.7 kb BamHI-EcoRV fragment using RecA-mediated single site cleavage 40 to cut uniquely at the BamHI site in exon 1 of the gene, before the ATG. The sequence of the oligo used to protect the BamHI site was as follows: 5′-CTCTGAAGGATAGAG-GATCCACTAAACAAAAACAAGGA-3 (BamHI site underlined).…”