Sequence-specific ligation of DNA using RecA protein

Abstract

A method is described that allows the sequence-specific ligation of DNA. The method is based on the ability of RecA protein from Escherichia coli to selectively pair oligonucleotides to their homologous sequences at the ends of fragments of duplex DNA. These three-stranded complexes were protected from the action of DNA polymerase. When treated with DNA polymerase, unprotected duplex fragments were converted to fragments with blunt ends, whereas protected fragments retained their cohesive ends. By using conditions that greatly favored ligation of cohesive ends, a second DNA fragment could be selectively ligated to a previously protected fragment of DNA. When this second DNA was a vector, selected fragments were preferentially cloned. The method had sufficient power to be used for the isolation of single-copy genes directly from yeast or human genomic DNA, and potentially could allow the isolation of much longer fragments with greater fidelity than obtainable by using PCR.In 1991, we described RecA-assisted restriction endonuclease (RARE) cleavage (1). The method was a general and efficient way to target restriction enzyme cleavage to unique, predetermined sites. The method utilized the ability of RecA protein to pair oligonucleotides to homologous sequences in duplex DNA to form three-stranded complexes. These complexes protected the selected sites from enzymatic methylation, and after dissociation of the complexes, restriction enzyme cleavage was limited to the selected unmethylated sites. We later improved the method to cleave fragments greater than a megabase in size (2), and it has been used by numerous investigators to map and manipulate large segments of DNA (3-18). We have now developed a complementary method that is functionally the reverse of RARE cleavage, in that the method uses RecA protein and oligonucleotides to direct the sequence-specific ligation of DNA. It represents the first report of targeting of the action of DNA ligase. When one of the DNA segments is a vector, it is possible to perform sequence-specific cloning of a selected genomic DNA segment. When used in this manner, we refer to the technique as RecA-assisted cloning (RAC).Several methods are available to amplify genomic DNA and to isolate selected fragments in a pure form. The most widely used method is PCR. A major limitation of PCR is the small size that may be reliably amplified, although improvements have allowed amplification of up to 20-30 kb by using human genomic DNA as the template (19-21). The other widely used general method to isolate genomic fragments involves constructing and screening DNA libraries. Libraries based on phage or cosmid vectors have long been in use, and several other vectors are now available for cloning large (Ͼ100 kb) segments of DNA in the form of yeast artificial chromosomes, bacterial artificial chromosomes, and P1 phage-derived artificial chromosomes. Such libraries, however, are tedious to construct and screen.Several groups have described strategies to use RecA protein from E. coli to scr...

Modulating cellular recombination potential through alterations in RecA structure and regulation

“…The well‐studied EcRecA protein has been used as a reagent in a wide range of applications in biotechnology [e.g. see (Ferrin and Camerini‐Otero, 1991; 1998; Szybalski, 1997; Zhumabayeva et al ., 1999; 2001; Wang et al ., 2006)]. Only a few RecA‐based technologies are widely used, and many attempts to use RecA for genomic engineering in various species have had limited success.…”

Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics

“…Numerous applications of RecA are reported. These include identification of different Burkholderia cepacia genomovars, 16 sequence-specific ligation of DNA using the E. coli RecA protein, 17 maintaining the integrity of chloroplast DNA molecules in Arabidopsis , 18 auto-cleavage of other serine proteases and repressors of the bacteriophage lytic cycle, 19 , 20 , 21 , 22 and its role in swarming motility in E. coli . 23 …”