Type II topoisomerases help regulate DNA topology during transcription, replication and recombination by catalysing DNA strand transfer through transient double-stranded breaks. All type II topoisomerases described so far are members of a single protein family. We have cloned and sequenced the genes encoding the A and B subunits of topoisomerase II from the archaeon Sulfolobus shibatae. This enzyme is the first of a new family. It has no similarity with other type II topoisomerases, except for three motifs in the B subunit probably involved in ATP binding and hydrolysis. We also found these motifs in proteins of the Hsp90 and MutL families. The A subunit has similarities with four proteins of unknown function. One of them, the Saccharomyces cerevisiae Spo11 protein, is required for the initiation of meiotic recombination. Mutagenesis, performed on SPO11, of the single tyrosine conserved between the five homologues shows that this amino acid is essential for Spo11 activity. By analogy with the mechanism of action of known type II topoisomerases, we suggest that Spo11 catalyses the formation of double-strand breaks that initiate meiotic recombination in S. cerevisiae.

Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a critical component of complexes of DSB-promoting proteins which assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution owing to reduced selectivity for sites that normally attract DSBs.Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes.Consequently, sex chromosomes do not form crossovers leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects are accompanied by a genome-wide delay in assembling DSB-promoting proteins on axes and a loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated complexes of DSB-promoting proteins. Highlights (85 characters max)Temporal and spatial patterning of recombination are regulated by ANKRD31 Selective use of PRDM9 binding sites as DSB hotspots requires ANKRD31 Enrichment of pro-DSB factors in the PAR requires ANKRD31 but not IHO1 Recombination in the PAR critically depends on ANKRD31 are consistent with unpublished data of Acquaviva, Jasin & Keeney (personal communication).Acquaviva et al. observed ANKRD31 aggregates on PARs, and identified chromosome 4, 9 and 13 as autosomes whose non-centromeric ends carry arrays of PAR-like sequences that associate with ANKRD31 aggregates. Distinct molecular requirements for ANKRD31 aggregates and ANKRD31 fociGiven their distinct behaviour in synapsed regions, foci and aggregates of ANKRD31/MEI4/REC114 might represent qualitatively different protein complexes with distinct underlying molecular requirements. To test this possibility we compared localization of ANKRD31, REC114 and MEI4 in Mei4 -/-, Rec114 -/and Iho1 -/spermatocytes ( Figure 2J and S3E-I). The numbers of REC114 and MEI4 foci are strongly reduced in Mei4 -/and Rec114 -/mice, respectively (Kumar et al., 2018). In addition, we found that both focus and aggregate formation of ANKRD31, REC114 and MEI4 were each disrupted in Mei4 -/and Rec114 -/spermatocytes ( Figure S3E-G and Table S3). Remarkably, only the formation of ANKRD31 ( Figure 2I), MEI4 and REC114 foci (Stanzione et al., 2016), but not aggregates, were disrupted in Iho1 -/spermatocytes ( Figure 2J). ANKRD31 aggregates formed efficiently in Iho1 -/spermatocytes; median numbers of ANKRD31 aggregates were four in both wild type (n=62) and Iho1 -/-(n=57) spermatocytes in zygotene. These aggregates always colocalized with aggregates of MEI4 (n=100) and REC114 (n=100) in Iho1 -/spermatocytes ( Figure 2J and S3H).ANKRD31 aggregates also colocalized with PAR FISH signals in late zygotene-like Iho1 -/spe...

Mitotic recombination within the ARG4 gene of Saccharomyces cerevisiae was analysed after treatment of cells with the recombinogenic agent 8-methoxypsoralen (8-MOP) plus UVA. The appearance of DNA double-strand breaks (DSBs) in the ARG4 region during post-treatment incubation was also tested. The results obtained after 8-MOP plus UVA treatment indicate that in mitotic cells: (1) recombination at the ARG4 locus is increased 30 - 500 fold per survivor depending on the strains and the doses employed, (2) the increase of recombination results essentially from gene conversion events which involve the RV site located in the 5' region of the ARG4 gene twice as often as the Bgl site at the 3' end, (3) depending on 8-MOP/UVA dose, ectopic gene conversion is associated with reciprocal translocation, (4) DSBs occur preferentially in the ARG 5' region during post-treatment incubation, as well as in other intergenic regions containing both promoters or/and terminators of transcription, and (5) changes in sequence content in the 5' region of ARG4, which influences positions and frequencies of DSBs formed during repair, are correlated with a modification of the local chromatin structure.