Bernd Reiss , Manfred Klemm , Hans Kosak , and Jeff Schell

A number of RecA-like proteins have been found in eukaryotic organisms. We demonstrate that the prokaryotic recombination protein RecA itself is capable of interacting with genomic homologous DNA in somatic plant cells. Resistance to the DNA crosslinking agent mitomycin C requires homologous recombination as well as excision repair activity. Tobacco protoplasts expressing a nucleus-targeted RecA protein were at least three times as efficient as wild-type cells in repairing mitomycin C-induced damage. Moreover, homologous recombination at a defined locus carrying an endogenous nuclear marker gene was stimulated at least 10-fold in transgenic plant cells expressing nucleus-targeted RecA. The increase in resistance to mitomycin C and the stimulation of intrachromosomal recombination demonstrate that Escherichia coli RecA protein is functional in genomic homologous recombination in plants, especially when targeted to the plant nucleus. The process of homologous recombination requires search for homology, recognition of sequence similarity, and strand exchange between two DNA molecules. In Escherichia coli these different steps are mediated by a single protein, the RecA protein (for review see ref. 1), which plays a central role in the recombination pathway of this bacterium. However, additional proteins are needed to initiate recombination and to resolve the intermediates created by RecA. Recombination is initiated by the generation of single-stranded DNA (ssDNA) and DNA ends in E. coli and presumably in all organisms. In E. coli, the combined action of the products of the recB, recC, and recD genes initiates a major recombination pathway (for review see ref. 2). ssDNA is recognized by RecA protein, and homologous double-stranded DNA is actively searched for. Exchange of complementary strands leads to the formation of recombination intermediates (Holliday structures). The intermediates can be resolved by different pathways; the major one involves the action of the RuvA, RuvB, and RuvC proteins. All of the recombination proteins have to work in concert to complete recombination successfully. A number of proteins with similarity to RecA have been found in eukaryotic cells such as budding yeast, fission yeast, humans, mice, chicken, and plants (ref. 3; references in ref. 4). The best-characterized ones are the Dmcl and Rad51 proteins from Saccharomyces cerevisiae. Both proteins show considerable sequence homology to RecA; in addition, Rad51 forms DNA/protein filaments, strikingly similar in tertiary structure to those formed with RecA (5). In addition to RecA-like proteins, a different class of strand-exchange proteins was found in eukaryotic cells (for review see ref. 4). The distinguishing feature of most of these proteins is that they do not require ATP to stimulate strand exchange. Rather than a search for homology and promotion of strand invasion, recombination involves exposure of ssDNA by exonuclease and subsequent reformation of double-stranded DNA from comThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. plementary ssDNAs. The mechanism is therefore inherently different from that of RecA-mediated recombination. Therefore, in eukaryotic cells, several RecA-like proteins seem to cooperate and another class of strand-exchange proteins might be needed at the same time. In light of the complexity of eukaryotic recombination the question arises whether true RecA-type recombination is possible and can be mediated by a single protein in a higher eukaryote such as a plant cell. To address this question, the key protein of the E. coli recombination pathway, RecA, was expressed in transgenic plants, and its effect on homologous recombination was analyzed in two different ways. Mitomycin C is known to intercalate in vivo into DNA, leading to crosslinking of complementary strands (6). Crosslinking leads to inhibition of DNA synthesis in bacteria without concomitant effects on RNA or protein synthesis (7). The data indicate that mitomycin C efficiently blocks DNA replication. The resultant daughter-strand blocks can be repaired by homologous recombination (sister-chromatid exchange) and excision repair. We found that high mitomycin C concentrations kill plant cells efficiently, presumably because the capacity of this repair/recombination system is exhausted. Plant cells expressing RecA, however, exhibited a considerably higher resistance to this drug. This suggests that RecA can function in plant cells, interacting with or supplementing the endogenous plant recombination machinery. Furthermore, RecA was shown to stimulate intrachromosomal recombination in plants directly. To be maximally effective, the E. coli RecA had to be targeted to the plant nucleus. This was achieved by expressing a RecA construct coding for a fusion protein of a nuclear targeting sequence from the large T antigen of simian virus 40 (SV40) and the RecA protein (nt-RecA) in transgenic tobacco cells. MATERIALS AND METHODS Plasmid Constructions. Modified recA genes were derived from plasmid pDR1453 (8). The plasmid was digested with the restriction enzyme Sac II, the ends were made blunt with DNA polymerase I large fragment, and the amino-terminusencoding part of the recA gene was subcloned as a Sac II/EcoRI fragment in plasmid pUC18, which had been cut with EcoRI and Sma I, yielding plasmid pRecA-1. The same plasmid was digested with Hinfl, the ends were rendered blunt, and the carboxyl-terminus-encoding part of the recA gene was subcloned in pUC19 (EcoRI/Sma I) as a HinflI/EcoRI fragment, yielding plasmid pRecA-2. The amino-terminusencoding part was further modified. A BstXI/EcoRI and a Taq I/BstXI fragment obtained from pRecA-1 encoding the amino-terminal part of recA without its initiation codon and two complementary oligonucleotides (5'-GGG GAC TCC TCC TAA GAA GAA GCG TAA GGT TAT GGC GAT-3' and 5'-CGA TCG CCA TAA CCT TAC GCT TCT TCT TAG Abbreviations: ssDNA, single-stranded DNA; SV40, simian virus 40; CaMV, cauliflower mosaic virus; Sulr, sulfonamide resistance. *To whom reprint requests should be addressed.