(NAS Colloquium) Links Between Recombination and Replication: Vital Roles of Recombination

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Colloquium on Links Between Recombination and Replication: Vital Roles of Recombination

Colloquium

Repeat expansion by homologous recombination in the mouse germ line at palindromic sequences

Zhi-Hong Zhou*, Ercan Akgün*, and Maria Jasin^†

Cell Biology Program, Sloan-Kettering Institute and Cornell University Graduate School of Medical Sciences, 1275 York Avenue, New York, NY 10021

Genetic instability can be induced by unusual DNA structures and sequence repeats. We have previously demonstrated that a large palindrome in the mouse germ line derived from transgene integration is extremely unstable and undergoes stabilizing rearrangements at high frequency, often through deletions that produce asymmetry. We have now characterized other palindrome rearrangements that arise from complex homologous recombination events. The structure of the recombinants is consistent with homologous recombination occurring by a noncrossover gene conversion mechanism in which a break induced in the palindrome promotes homologous strand invasion and repair synthesis, similar to mitotic break repair events reported in mammalian cells. Some of the homologous recombination events led to expansion in the size of the palindromic locus, which in the extreme case more than doubled the number of repeats. These results may have implications for instability observed at naturally occurring palindromic or quasipalindromic sequences.

palindrome | hairpin | sequence repeats | sister chromatid recombination

Perfect palindromes and inverted repeats separated by spacers, both of which have the potential to form secondary structures, manifest genetic instability in Escherichia coli (1), yeast (2), and in the mouse (3, 4). As a result, palindromes and inverted repeats are considered “at-risk motifs” for the genome (2). The degree of instability associated with palindromes and inverted repeats depends on several factors, such as the repeat length, the distance between repeats, and the homology shared by the repeats (1, 5). A history of inverted repeat instability has been suggested during the evolution of the human genome by the analysis of the distribution of Alu elements. The superabundance and dense clustering of Alu repeats predicts that nearby inverted Alu elements would be frequent in the genome. However, closely spaced highly homologous inverted Alu repeats are substantially underrepresented relative to direct repeats in regions able to be sequenced, as if they may have been removed from the genome (5, 6).

In E. coli, long palindromes confer inviability to replicons, presumably because of the induction of strand breaks that lead to degradation of the replicon (1). In eukaryotes, long palindromes can be propagated, but they nevertheless manifest instability. For example, in yeast, a 2-kb palindrome stimulates intra- and interchromosomal homologous recombination three and four orders of magnitude, respectively, and is also deleted at high frequency (7). A much smaller palindrome of 140 bp has been shown to create meiotic recombination hotspots by generating sites for double-strand breaks (DSBs) (8).

In an earlier report, we demonstrated that a long palindrome in the mouse germ line manifests a high degree of instability. The previously characterized events were primarily nonhomologous deletions at the center of symmetry, although we had also observed frequent gene conversion events within direct repeats contained in the palindrome by using a β-galactosidase sperm assay (4, 9). We now show that other outcomes of homologous recombination occur at high frequency at repeat sequences within or adjacent to the palindrome, approaching 50% in some mice. In particular, recombination events can result in repeat expansion, substantially increasing the size of the palindromic locus. The expansion events are most easily explained by a gene conversion mechanism that does not involve reciprocal exchange.

Materials and Methods

Transgenic Mice and Sperm Analysis. The derivations of mouse lines 78 and 2275 have been previously described (4). Mouse lines were maintained by breeding with (C57BL/6×CBA/Ca)F1 mice, and transgenic mice were identified by Southern blot analysis of tail-tip DNA by using the entire lacZ gene as probe.

Sperm preparations were similar to those previously described (4). Sperm were squeezed from the caudal epididymis of adult males into a PBS solution (PBS with 10 mM Hepes and 1% BSA). Sperm (2×10⁶) were placed in 0.5 ml of PBS solution, washed twice, and then resuspended in 0.2 ml of PBS solution. For staining, 20 µl (2×10⁵) of sperm was added to 30 µl of the PBS solution, and then 50 µl of 1 mM 5-chloromethylfluorescein di-β-D-galactopyranoside (CM-FDG; Molecular Probes) was added. After 60 sec, 1 ml of PBS solution was added. Sperm were analyzed on a FACScan (Becton Dickinson) after 30 min.

Results

Gene Conversion Within the Line 78 Palindromic Transgene. We have previously introduced a gene conversion reporter substrate into the mouse germ line that, on integration, formed a large palindrome (4). The palindrome, designated 78 for the number of the founder mouse, consists of two complete copies of the reporter substrate (78 parent; Fig. 1A). Each copy is 7.65 kb and is comprised of two defective lacZ genes, lacZ4∆, which contains a 4-bp deletion within an otherwise intact lacZ gene and in lacZ, which is a 1.8-kb internal lacZ fragment. In addition to creating a frameshift, the 4-bp deletion in lacZ4∆ disrupts a SacI site. A simple gene conversion (SGC) involving the lacZ4∆ and in lacZ genes can restore the sequence at the SacI site to create a lacZ⁺ gene (78 lacZ⁺; Fig. 1A). Because the promoter for the lacZ4∆ gene is derived from the spermatid-specific protamine 1 (Prm-1) gene, lacZ⁺ expression is detected postmeiotically by assaying β-galactosidase activity in spermatids or sperm.

The parental line 78 transgene locus was transmitted in a Mendelian fashion but rearranged at high frequency (4). Approximately 15–30% of progeny from mice with the palindrome had variant arrangements that frequently involved deletions at

This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA.

Abbreviations: DSB, double-strand break, SGC, simple gene conversion; CGC, complex gene conversion; SCE, sister chromatid exchange.

*	Z.-H.Z. and E.A. contributed equally to this work.
^†	To whom reprint requests should be addressed. E-mail: m-jasin@ski.mskcc.org.

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