Inverted repeats (IRs) that can form a hairpin or cruciform structures are common in the human genome and may be sources of chromosome instability. We have taken a broad approach to study their potential for instability and relate this to this incidence in the human genome. Our studies revealed a paucity of closely spaced diverged Irs. We have been studying an IR involving the human Alu sequence (Alu-IR), the most abundant inverted repeat in the human genome, as a model of such structures in the yeast Saccharomyces cerevisiae. We also chose Alus because they are identified with many human diseases and are often seen at chromosomal breakpoints in cancer cells. We showed that a quasipalindrome comprised of two closely-spaced inverted Alu sequences is highly unstable in yeast. The Alu-IR caused a 1000-fold increase in homologous ectopic recombination between lys2 genes. We established that the ability of an Alu-IR to cause genome rearrangements results from a novel type of double-strand break in mitotic cells. Using an approach developed in this lab for analyzing rare mitotic chromosomal breaks, we demonstrated that resolution of a secondary structure formed by IRs results in broken molecules that are capped by a hairpin. We found that the requirements for repair of hairpin-capped DSBs were more extensive than for other DSBs, suggesting that they might be more threatening to the genome than other types of breaks. In addition to the standard DSB repair genes, repair of IR-induced breaks depended on the endonuclease activity of Mre11/Rad50/Xrs2 complex and on Sae2 protein. Failure of the mre11, rad50, xrs2 and sae2 mutants to open and process the hairpins led to the generation of chromosome inverted duplications. Based on these results we proposed a new role for the Mre11 complex in maintaining genome stability, namely in protecting the genome against hairpins. The role of Mre11 complex might be especially crucial in preserving the integrity of complex genomes, particularly the human genome, that contain a large amount of repetitive DNA capable of forming secondary structures. The system that we developed is now being expanded to address the impact of DNA double-strand breaks on the stimulation of changes at quasipalindromes. We are creating at DNA double-strand break target site next to inverted repeat pairs of Alus of varying levels of divergence. This system will enable us to investigate the consequences of a break in a region near large inverted repeats of the sort that are common in the human genome. With this approach we will also address the impact of a break at a large distance from an inverted repeat as a model for what might happen in a genome with many inverted repeats. For example, this would mimic the situation for damage at a distance from an IR. Since resection can be quite large (many KB), a break can essentially telegraph the formation of an inverted chromosome duplication. We will also study the role/impact of mismatch repair when the Alus are diverged. These structures will be studied in G1 and G2 cells in wild type and various mutants to address genetic controls.
| Lobachev, Kirill S; Gordenin, Dmitry A; Resnick, Michael A (2002) The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements. Cell 108:183-93 |
| Stenger, J E; Lobachev, K S; Gordenin, D et al. (2001) Biased distribution of inverted and direct Alus in the human genome: implications for insertion, exclusion, and genome stability. Genome Res 11:12-27 |
| Lobachev, K S; Stenger, J E; Kozyreva, O G et al. (2000) Inverted Alu repeats unstable in yeast are excluded from the human genome. EMBO J 19:3822-30 |
