Palindrome Resolution and Recombination in the Mammalian Germ Line

Abstract

Genetic instability is promoted by unusual sequence arrangements and DNA structures. Hairpin DNA structures can form from palindromes and from triplet repeats, and they are also intermediates in V(D)J recombination. We have measured the genetic stability of a large palindrome which has the potential to form a one-stranded hairpin or a two-stranded cruciform structure and have analyzed recombinants at the molecular level. A palindrome of 15.3 kb introduced as a transgene was found to be transmitted at a normal Mendelian ratio in mice, in striking contrast to the profound instability of large palindromes in prokaryotic systems. In a significant number of progeny mice, however, the palindromic transgene is rearranged; between 15 and 56% of progeny contain rearrangements. Rearrangements within the palindromic repeat occur both by illegitimate and homologous, reciprocal recombination. Gene conversion within the transgene locus, as quantitated by a novel sperm fluorescence assay, is also elevated. Illegitimate events often take the form of an asymmetric deletion that eliminates the central symmetry of the palindrome. Such asymmetric transgene deletions, including those that maintain one complete half of the palindromic repeat, are stabilized so that they cannot undergo further illegitimate rearrangements, and they also exhibit reduced levels of gene conversion. By contrast, transgene rearrangements that maintain the central symmetry continue to be unstable. Based on the observed events, we propose that one mechanism promoting the instability of the palindrome may involve breaks generated at the hairpin structure by a hairpin-nicking activity, as previously detected in somatic cells. Because mammalian cells are capable of efficiently repairing chromosome breaks through nonhomologous processes, the resealing of such breaks introduces a stabilizing asymmetry at the center of the palindrome. We propose that the ability of mammalian cells to eliminate the perfect symmetry in a palindromic sequence may be an important DNA repair pathway, with implications regarding the metabolism of palindromic repeats, the mutability of quasipalindromic triplet repeats, and the early steps in gene amplification events.Recombination rates are known to vary along chromosomes. A fixed genetic distance can correspond to widely different physical lengths depending on the location on the chromosome. Although certain sequences such as the site in phage and Escherichia coli are recognized by enzymes that promote recombination (43), growing evidence suggests that DNA structure and accessibility, rather than sequence per se, have a profound impact on recombination. During meiosis in mammals, for example, very different recombination rates can be observed at identical chromosomal regions in males and females (11; for a review, see reference 45). In Saccharomyces cerevisiae, where most, if not all, meiotic recombination is initiated by double-strand breaks (48), DNA sequence is not the primary determinant of the position of breaks; rather, breaks map ...

Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase

“…Recombination reporters consisting of repeat sequences have been used to interrogate spontaneous and DNA damage-induced homologous recombination in both the soma and germ line of mice, but these events are generally rare, except in the case where the reporter is genetically unstable (35)(36)(37)(38). Moreover, interanimal variation can be high, requiring the use of large numbers of mice (∼50) to compare mutants (39).…”

Palindromic AT-rich repeat in theNF1gene is hypervariable in humans and evolutionarily conserved in primates

“…In bacterial and eukaryotic genomes, long palindromes are unstable and adopt secondary structures that appear to promote deletion [Leach, 1994;Collick et al, 1996;Akgün et al, 1997;Waldman et al, 1999;Cunningham et al, 2003]. In a eukaryotic genome, transgenes that form large inverted repeats are unstable in both mitosis and meiosis, leading to rearrangement or complete loss of the transgene [Collick et al, 1996].…”

Biased Distribution of Inverted and Direct Alus in the Human Genome: Implications for Insertion, Exclusion, and Genome Stability

“…Pairs of large inverted DNA repeats can be unstable, lead to deletions, and stimulate recombination between DNAs surrounding the inverted repeats in yeast (Gordenin et al 1993;Ruskin and Fink 1993;Nag and Kurst 1997;Tran et al 1997;Lobachev et al 1998Lobachev et al , 2000 ) and mammalian cells (Akgun et al 1997;Lewis 1999;Lewis et al 1999). Sequence identity was suggested as a likely factor in the stability of Alu repeats based on results in yeast.…”