Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by the evolutionarily conserved protein, Spo11 (1)
. Spo11 shares sequence similarity to subunit A of the type-II topoisomerase from archaea, Topo VI, and is thought to perform DSB catalysis in a similar way. Specifically, Spo11 dimerization creates two intermolecular DNA cleavage sites each consisting of a Mg2+
coordination site (Toprim domain) and the 5Y-CAP domain where the catalytic tyrosine residue resides. Concerted DNA strand breakage creates a DSB with a two-nucleotide 5′ overhang with the 5′ ends covalently linked to the catalytic tyrosine present in the active site of each monomer (residue 135 in yeast). Mutation of this residue, or indeed others within the active site, abolishes or reduces DSB formation (2, 3)
Topo VI is an A2
heterotetramer, which functions in isolation. Spo11, on the other hand, requires at least nine other proteins of unknown stoichiometry to support DSB catalysis (1)
. Most of these proteins have a poorly understood role in the catalytic process, and show poor evolutionary conservation (
. Nevertheless, a pattern of essential interactions between them is slowly emerging (4-6)
. Given that Spo11 shares homology with only the A subunit of Topo VI, it is possible that one or more of these proteins functions as an equivalent or substitute of the B subunit of Topo VI, which contains an essential ATPase domain.
In contrast to the transient lesions created by type II topoisomerases, Spo11-DSBs are not resealed by a reversal of the DNA cleavage reaction. Instead, Spo11-DSBs are channeled into a DNA repair pathway that involves 5′→3′ single-stranded exonucleolytic resection, DNA strand invasion and extension of the exposed single stranded ends, and finally Holliday junction resolution to yield intact, and recombinant, DNA molecules (7)
. The first step in this processing pathway is the removal of the covalently bound Spo11 protein from the DSB end, a process that requires the Mre11 complex and the accessory protein, Sae2 (1)
In cell lysates from both yeast and mouse, a fraction of Spo11 protein is detected covalently attached to short oligos that end with free 3′ OH termini (8)
. Detection of these Spo11-oligo complexes is dependent on meiotic DSB formation and on the activity of the Mre11 complex and Sae2, mutation in either of which prevents release of Spo11 from DSB ends. In both organisms, two forms of the Spo11-oligo complexes are detected, differing in the length of the attached DNA. In yeast, the shorter form has oligos of less than 12 nt, whereas the longer form has oligos between 21 and 37 nt in length. In mouse, the length distribution of the shorter oligos is more heterogeneous (12-26 nt), whereas the larger form is more discrete (28-34 nt) (8)
. In yeast, the two forms of Spo11-oligo complex are equally abundant, with kinetics of appearance and disappearance closely matching those of resected DSB molecules. Together these observations provide compelling evidence to support a model in which Spo11 is released from DSB ends via single-strand scissions flanking the DSB, a reaction that may be catalyzed by the Mre11 nuclease in combination with Sae2 (8)
The relationship between the long and short forms of Spo11-oligo complex is not known. However, one idea, inspired by the observation that the two forms are equally abundant, is that the nicks are positioned asymmetrically flanking the DSB, thus yielding one longer and one shorter Spo11-oligo complex at every DSB (). Moreover, if the DSB end is duplex at the time when the releasing nicks are created, differential stability of the Spo11-oligo complexes on either side of the DSB (due to the extent of DNA base pairing) could create one free DSB end and one DSB end blocked by the Spo11-oligo complexes. The lifespan of the Spo11-oligo complexes might then be expected to match that of unrepaired DSBs, as is observed. Direct physical evidence to support such a model is being sought.
Fig 1 Endonucleolytic release of Spo11 from DSB ends. Spo11 catalyses DSB formation, becoming covalently attached to the 5′ ends at the break site. Single-stranded endonucleolytic cleavage flanking the Spo11-DSB permits release of Spo11 that is still (more ...)
Here, we present a method for detection of Spo11-oligo complexes from yeast (see ). The methodology involves purifying Spo11 complexes from cell lysates by immunoprecipitation, followed by end-labeling any copurifying free 3′ OH DNA ends with radiolabeled nucleotide by using the template-independent polymerase, terminal deoxynucleotidyl transferase (TdT). End-labeled Spo11-oligo complexes are separated from any end-labeled DNA contaminants by fractionation on SDS-PAGE, and are detected by autoradiography. We prepare yeast lysates by breaking cells in the presence of the strong denaturant, trichloroacetic acid (TCA), and dissolving the resulting precipitate in Tris-buffered SDS. This method minimizes proteolysis, which is otherwise a significant problem when preparing meiotic protein extracts. However, care should be taken to keep temperatures low at all times, since the strongly acidic conditions can lead to reduction in Spo11-oligo labeling yield most likely due to acid-induced DNA depurination. The relative abundance of the Spo11-oligos appears to be a good measure of the relative frequency of DSB formation within the cell. In principle, we believe that with suitable modification such methods could also be used to detect Spo11-oligo complexes in other organisms, and would thus provide a long-sought biochemical assay to measure DSB frequency.
Fig 2 Scheme for detecting Spo11-oligo complexes from meiotic yeast cells. A) Meiotic cells are harvested and lysed in 10% TCA with zirconia beads. Precipitated protein in the extract is dissolved with 2% SDS and soluble protein diluted with Triton X100. Spo11-HA3His6 (more ...)