Fission yeast hotspot sequence motifs are also active in budding yeast.

In most organisms, including humans, meiotic recombination occurs preferentially at a limited number of sites in the genome known as hotspots. There has been substantial progress recently in elucidating the factors determining the location of meiotic recombination hotspots, and it is becoming clear that simple sequence motifs play a significant role. In S. pombe, there are at least five unique sequence motifs that have been shown to produce hotspots of recombination, and it is likely that there are more. In S. cerevisiae, simple sequence motifs have also been shown to produce hotspots or show significant correlations with hotspots. Some of the hotspot motifs in both yeasts are known or suspected to bind transcription factors (TFs), which are required for the activity of those hotspots. Here we show that four of the five hotspot motifs identified in S. pombe also create hotspots in the distantly related budding yeast S. cerevisiae. For one of these hotspots, M26 (also called CRE), we identify TFs, Cst6 and Sko1, that activate and inhibit the hotspot, respectively. In addition, two of the hotspot motifs show significant correlations with naturally occurring hotspots. The conservation of these hotspots between the distantly related fission and budding yeasts suggests that these sequence motifs, and others yet to be discovered, may function widely as hotspots in many diverse organisms.

Novel nucleotide sequence motifs that produce hotspots of meiotic recombination in Schizosaccharomyces pombe.

In many organisms, including yeasts and humans, meiotic recombination is initiated preferentially at a limited number of sites in the genome referred to as recombination hotspots. Predicting precisely the location of most hotspots has remained elusive. In this study, we tested the hypothesis that hotspots can result from multiple different sequence motifs. We devised a method to rapidly screen many short random oligonucleotide sequences for hotspot activity in the fission yeast Schizosaccharomyces pombe and produced a library of approximately 500 unique 15- and 30-bp sequences containing hotspots. The frequency of hotspots found suggests that there may be a relatively large number of different sequence motifs that produce hotspots. Within our sequence library, we found many shorter 6- to 10-bp motifs that occurred multiple times, many of which produced hotspots when reconstructed in vivo. On the basis of sequence similarity, we were able to group those hotspots into five different sequence families. At least one of the novel hotspots we found appears to be a target for a transcription factor, as it requires that factor for its hotspot activity. We propose that many hotspots in S. pombe, and perhaps other organisms, result from simple sequence motifs, some of which are identified here.

Mps1 activation loop autophosphorylation enhances kinase activity.

The Mps1 protein kinase is required for proper assembly of the mitotic spindle, checkpoint signaling, and several other aspects of cell growth and differentiation. Mps1 regulation is mediated by cell cycle-dependent changes in transcription and protein level. There is also a strong correlation between hyperphosphorylated mitotic forms of Mps1 and increased kinase activity. We investigated the role that autophosphorylation plays in regulating human Mps1 (hMps1) protein kinase activity. Here we report that hyperphosphorylated hMps1 forms are not the only active forms of the kinase. However, autophosphorylation of hMps1 within the activation loop is required for full activity in vitro. Mass spectrometry analysis of de novo synthesized enzyme in Escherichia coli identified autophosphorylation sites at residues Thr(675), Thr(676), and Thr(686), but phosphatase-treated and reactivated enzyme was only phosphorylated on Thr(676). Mutation of Thr(676) in hMps1 or the corresponding Thr(591) residue within yeast Mps1 reduces kinase activity in vitro. We find that overexpression of an hMps1-T676A mutation inhibits centrosome duplication in RPE1 cells. Likewise, yeast cells harboring mps1-T591A as the sole MPS1 allele are not viable. Our data strongly support the conclusion that site-specific Mps1 autophosphorylation within the activation loop is required for full activity in vitro and function in vivo.