Homologous Recombination in Higher Plants: Clues from fasciata1-4, a New Chromatin Formation Mutant of Arabidopsis

Genetic recombination is one of the most fundamental and significant events in the history of life because it plays a central role in creating genetic diversity and safeguarding and maintaining genomic integrity. Homologous recombination (HR) was first described by Thomas Hunt Morgan (Morgan, 1916) and results from crossingover of paired non-sister chromatids during prophase I of meiosis (see figure). HR has been extensively studied, and the functions of many of the key factors are well understood in a number of model organisms. All students of biology are familiar with the basic steps, which result in an exchange of information generating daughter chromosomes that comprise unique combinations of genetic loci. Any pair of homologous chromosomes may be expected to cross over multiple times during meiosis, and more advanced students will also be familiar with how this seemingly simple process becomes more and more complicated the more it is studied. In fact, some essential aspects of HR remain murky. For example, although HR has been exploited for targeted gene replacement in a number of model organisms, such as yeast, mice, and Drosophila, higher plants remain largely recalcitrant to this approach, owing to low efficiencies of HR and poor reproducibility for reasons that are not well understood (reviewed in Hanin and Paszkowski, 2003; Puchta, 2003; Reiss, 2003). Targeted gene replacement is an exceptionally powerful tool that allows in-depth analysis of gene function through the creation of highly specific alleles. Transformation vectors are designed to facilitate HR and selection of recombinant progeny, resulting in genomic replacement of an endogenous gene with an in vitro–modified copy. Winzeler et al. (1999) used this approach to analyze the functions of nearly all of the 6200 open reading frames of the yeast Saccharomyces cerevisiae, and it is routinely used to investigate gene function in mice and Drosophila. Gene targeting has enormous potential for the precise engineering of transgenes for commercial agronomic and therapeutic applications, and its application in higher plants would be a most welcome addition to plant genomics. Hanin et al. (2001) and Terada et al. (2002) reported relatively high efficiencies of gene targeting using HR in Arabidopsis and rice, respectively, but these reports did not lead to an immediate breakthrough in the technology, and reproducibility in both systems remained relatively low. Recently, a few more reports of successful gene targeting in plants have appeared, indicating that the technology is improving. Shaked et al. (2005) found that expression of the yeast RAD54 chromatin remodeling gene enhanced HR in Arabidopsis by one to two orders of magnitude, providing evidence that chromatin conformation is a factor restricting HR in plants. It is also known that HR can be enhanced by the creation of double-strand breaks at the target site (Puchta et al., 1996), and Wright et al. (2005) demonstrated the feasibility of designing zinc-finger nucleases to induce double-strand breaks and enhance HR at specific target sites in plants. Nonetheless, a deeper understanding of factors controlling and restricting HR in plants is needed. Observations of high efficiencies of extrachromosomal HR in plants (which occurs, for example, in engineered recombination systems and between T-DNAs during Agrobacterium-mediated transformation), but routinely low efficiencies of chromosomal HR, have long suggested that chromatin might play a role in restricting HR in plant systems (reviewed in Reiss, 2003). In this issue of The Plant Cell, Kirik et al. (pages 2431–2442) provide further evidence that chromatin conformation is a major factor restricting HR in higher plants, through analysis of an Arabidopsis mutant with defects in chromatin assembly factor 1 (CAF-1), a heterotrimeric complex that is required for in vitro nucleosome assembly onto newly replicated chromatin in eukaryotic systems. The authors isolated a new allele of FASCIATA1 (FAS1), which encodes the p150 subunit of CAF-1. Plants containing the fas1-4 allele exhibit a severe developmental phenotype and reduced heterochromatin content compared with the wild type, along with a more open conformation of euchromatin, and, significantly, an ;100-fold enhanced rate of intrachromosomal HR, which is by far the strongest effect on intrachromosomal HR of all chromatin mutants analyzed in plants thus far. These results, together with nearly normal expression of several known HR genes in the fas1-4 mutant, suggest that chromatin conformation is a key factor limiting HR in plants. CAF-1 is evolutionarily conserved, and genes encoding the three subunits, p150, p60, and p48, are present in yeast and human cells as well as in plants. In human cells, CAF-1 interacts directly with proliferating cell nuclear antigen and functions to tether this complex to the growing replication fork during DNA replication. CAF-1 function has been linked to DNA