Analysis of intrachromosomal homologous recombination in mammalian cell, using tandem repeat sequences

Abstract

In all the organisms, homologous recombination (HR) is involved in fundamental processes such as genome diversification and DNA repair. Several strategies can be devised to measure homologous recombination in mammalian cells. We present here the interest of using intrachromosomal tandem repeat sequences to measure HR in mammalian cells and we discuss the differences with the ectopic plasmids recombination. The present review focuses on the molecular mechanisms of HR between tandem repeats in mammalian cells. The possibility to use two different orientations of tandem repeats (direct or inverted repeats) in parallel constitutes also an advantage. While inverted repeats measure only events arising by strand exchange (gene conversion and crossing over), direct repeats monitor strand exchange events and also non-conservative processes such as single strand annealing or replication slippage. In yeast, these processes depend on different pathways, most of them also existing in mammalian cells. These data permit to devise substrates adapted to specific questions about HR in mammalian cells. The effect of substrate structures (heterologies, insertions/deletions, GT repeats, transcription) and consequences of DNA double strand breaks induced by ionizing radiation or endonuclease (especially the rare-cutting endonuclease ISce-I) on HR are discussed. Finally, transgenic mouse models using tandem repeats are briefly presented.

Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice

“…Most of the time, sequences are aligned perfectly, and flanking sequences are not exchanged. However, misalignments may result in deletions, and exchanges between homologous chromosomes may lead to loss of heterozygosity, events that are known to promote cancer (11)(12)(13)(14).A useful approach to studying recombination is to engineer two mutant expression cassettes in a direct repeat and assay for restoration of wild-type gene expression in cultured cells (2,15,16). For example, by using direct-repeat substrates, it has been shown that homologous recombination repairs 30-50% of double-strand breaks in mammalian cells (17).…”

“…A useful approach to studying recombination is to engineer two mutant expression cassettes in a direct repeat and assay for restoration of wild-type gene expression in cultured cells (2,15,16). For example, by using direct-repeat substrates, it has been shown that homologous recombination repairs 30-50% of double-strand breaks in mammalian cells (17).…”

Genomic Structure of the Mouse Ap3b1 Gene in Normal and Pearl Mice

“…Though two partial Alu-like sequences were found in the 5Ј and 3Ј introns (Fig. 3), they are in opposite orientation and thus cannot produce a tandem duplication by the usual homologous recombination models (Hu and Worton, 1992;Purandare and Patel, 1997;Lambert et al, 1999). No other significant homologies were found at the breakpoints of 5Ј or 3Ј introns or the junction points.…”

“…Tandem duplications involving a portion of a single gene have been reported for a number of genes (Hu and Worton, 1992;Deininger and Batzer, 1999). Two general mechanisms leading to such tandem duplications have been proposed: homologous and nonhomologous recombination (Hu and Worton, 1992;Purandare and Patel, 1997;Lambert et al, 1999). Homologous recombination occurs between a stretch of homologous sequences, like Alu repeats (Gondo et al, 1993;Deininger and Batzer, 1999;Lambert et al, 1999).…”

“…Two general mechanisms leading to such tandem duplications have been proposed: homologous and nonhomologous recombination (Hu and Worton, 1992;Purandare and Patel, 1997;Lambert et al, 1999). Homologous recombination occurs between a stretch of homologous sequences, like Alu repeats (Gondo et al, 1993;Deininger and Batzer, 1999;Lambert et al, 1999). In these cases, the breakpoints and junction points are usually located within homologous sequences.…”