RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium

Abstract

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of doublestrand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.T he RecA protein plays a central role in the homologous recombination pathway of Escherichia coli. RecA alone mediates the search for homology, recognition of sequence similarities, and strand exchange between two DNA molecules. Homologues of this protein have been found in bacteria and archea as well as in lower and higher eukaryotes. The best known RecA homologues are the eukaryotic Rad51 proteins. Biochemical analysis of these proteins demonstrated that they are not only structural but also functional RecA homologues (reviewed in refs. 1-3).The bacterial protein RecA protein and a derivative carrying a nuclear localization signal (nt-RecA, ref. 4) have been expressed in tobacco plants and shown to stimulate homologous recombination in plants by using intrachromosomal recombination and DNAdamage repair assays (5). Studies in a mammalian system suggest that ectopic expression of the Rad51 protein can have a similar effect (6, 7). A key player in the E. coli recombination pathway is also the Holliday junction resolvase RuvC. Overexpression of this protein also enhances homologous recombination in plants (8). These data suggest that ectopic expression of several components of the homologous recombination pathway can stimulate the homologous recombination reaction.Another important aspect determining the overall efficiency of recombination is initiation. Induction of site-specific doublestrand breaks (DSBs) has been shown to stimulate homologous recombination at the target locus by several orders of magnitude in eukaryotes (for review, see refs. 9, 10). It has been demonstrated that homologous recombination is a significant DSB repair pathway in plants and that both ends of the break are processed independently and in a step-wise fashion (refs. 11-16; for review, see ref. 17). Thus, products with either one (''onesided'') or two (''two-sided'') homologous junctions can arise during the reaction.Although several recombination enzymes have been ...

Targeted transgene integration in plant cells using designed zinc finger nucleases

“…Site-specific transgene integration occurs at a very low frequency in plant cells as compared to random integration, even when the introduced DNA contains large stretches of sequence with homology to host DNA (Paszkowski et al 1988;Offringa et al 1990;Lee et al 1990;Miao and Lam 1995;Kempin et al 1997;Hanin et al 2001). Attempts to enhance targeted integration efficiencies in plants have included the use of negative selection markers (Wang et al 2001;Terada et al 2002Terada et al , 2007 and plants genetically engineered to exhibit modified recombination processes (Shalev et al 1999;Reiss et al 2000). These efforts notwithstanding, site-specific transgene integration in plant cells remains a challenge (Puchta 2005).…”

Enhancing gene targeting efficiency in higher plants: rice is on the move

“…However, Rad52 homologs have been discovered in animals, which are known to perform GT at low levels with the exception of some characterised cell-lines (see above). Moreover, homologs of most other proteins involved in HR have been cloned in plants and somatic GT events do occur despite the absence of Rad52 (Paszkowski et al, 1988;Lee et al, 1990;Offringa et al, 1990;Miao & Lam, 1995;Risseeuw et al, 1995Risseeuw et al, , 1997Kempin et al, 1997;Reiss et al, 2000;Hanin et al, 2001;Terada et al, 2002). It is also possible that Rad52 function is accomplished by an unknown component in plants and animals.…”

“…The consequence of the preferential DSB repair pathway in higher plants is that natural frequencies of GT by HR remain very low, ranging from 10 )3 to 10 )6 (Paszkowski et al, 1988;Lee et al, 1990;Offringa et al, 1990;Miao & Lam, 1995;Risseeuw et al, 1995Risseeuw et al, , 1997Kempin et al, 1997;Reiss et al, 2000;Hanin et al, 2001;Terada et al, 2002). Thus, the main question to address is why the recombination machinery is so efficient in some organisms, such as yeast or the moss Physcomitrella patens, but not in higher plants.…”

“…In higher plants, since the first report of targeted reconstruction of a transgene in the tobacco genome (Paszkowski et al, 1988), GT events have been described at several loci but only in dicotyledonous plants and always with a very low frequency ranging from 10 )3 to 10 )6 (Lee et al, 1990;Offringa et al, 1990;Miao & Lam, 1995;Risseeuw et al, 1995Risseeuw et al, , 1997Kempin et al, 1997;Reiss et al, 2000;Hanin et al, 2001). However, a recent report described an efficient method of T-DNA-mediated targeted disruption of a non-selectable locus (the waxy gene) in rice based on a large homologous region of 13 kb and highly expressed, duplicated diphtheria toxin gene-based counter-selectable markers (Terada et al, 2002;reviewed by lida & Terada, 2004).…”

Thermostable lichenase as a translational reporter

“…2c). Earlier, it was shown that the E. coli RecA protein stimulates homologous recombination in plants [17,18]. This indicates that the expression of hybrid gene recAlicBM2 in plants also can alter the recombination rate and spectrum, ensuring the use of more simple and sensitive methods to analyze the hybrid gene expression.…”