Abstract
Meiotic recombination is initiated by DNA double-stranded break (DSB) formation catalyzed by Spo11, a type-II topoisomerase-like transesterificase, presumably via a dimerization-mediated mechanism. We demonstrate the existence of in vivo interactions between Spo11 proteins carrying distinct tags, and the chromatin-binding and DSB activity of tagged Spo11 at innate and targeted DSB sites upon fusion to the Gal4 DNA-binding domain. First we identified the interaction between Spo11-3FLAG and Gal4BD-Spo11 proteins, and established that this interaction specifically occurs at the time of DSB formation. We then observed that presence of the Gal4BD-spo11Y135F (nuclease-deficient) protein allows Spo11-3FLAG recruitment at the GAL2 locus, indicative of the formation of a hetero-complex near the GAL2 UAS sites, but no formation of double- or single-strand breaks. Spo11 self-interaction around the GAL2 DSB site depends on other proteins for DSB formation, in particular Rec102, Rec104 and Rec114. Together, these results suggest that in vivo self-association of Spo11 during meiosis is genetically regulated. The results are discussed in relation to possible roles of Spo11 self-interaction in the control of the cleavage activity.
| Original language | English |
|---|---|
| Pages (from-to) | 1119-1133 |
| Journal | Nucleic Acids Research |
| Volume | 35 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 2007 |
Bibliographical note
AcknowledgementsWe thank K. Hirota and K. Kugou for their help, advice and comments; and H. Seo for critical discussion regarding the Gal4BD-spo11Y135F/Spo11-3FLAG in vivo assay. We are also grateful to other laboratory members for helpful discussions. A.N. thanks RIKEN and T. Shibata for support from the RIKEN eminent scientist program. This work was supported by grants for basic research from the Bio-oriented Technology Research Advancement Institution (to K.O. and T.S.) and grants-in-aid for scientific research on priority areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Funding to pay the Open Access publication charge was provided by the grants listed above.