Chromosomal context dependence of a eukaryotic recombinational hot spot.

Abstract

The single base-pair mutation M26 in the ade6 gene of the fiion yeast Schizosaccharomyces pombe creates a hot spot for meiotic homologous recombination. When DNA fragments containing M26 and up to 3.0 kilobases of surrounding DNA were moved to the ura4 gene or to a multicopy plasmid, M26 had no detectable hot spot activity.Our results indicate that nucleotide sequences at least 1 kilobase away from M26 are required for M26 hot spot activity and suggest that, as for transcriptional promoters, a second site or proper chromatin structure is required for activation of this eukaryotic recombinational hot spot. We discuss the implications of these results for studies of other meiotic recombinational hot spots and for gene targeting.Special sites enhance homologous recombination within their vicinity on the chromosomes ofprokaryotes and eukaryotes (1). These sites, called hot spots, are thought to be recognition sites for recombination-promoting enzymes, which act at high frequency at or near the hot spot. Recombinational hot spots are thus analogous to promoters of transcription and origins of DNA replication, in that they define discrete chromosomal sites that stimulate chromosomal activity in their vicinity.The M26 mutation in the ade6 gene of Schizosaccharomyces pombe creates a recombinational hot spot (2) that enhances gene conversion and crossing-over at the ade6 locus but not at other loci; the M26 hot spot is active during meiosis but not during mitosis (3, 4). In some respects the M26 hot spot appears simple: it is a single base-pair change G-C -* T-A (3, 5) creating the sequence 5'-ATGACGT-3', which is required for hot spot activity (6). M26 acts when it is present on one or both of the recombining chromosomes (3). These results are consistent with the view that M26 is the recognition site for a sequence-specific recombination-promoting protein. Indeed, an M26-specific binding protein has recently been detected (E. Kaslin and J. Kohli, personal communication; W. Wahis and G.R.S., unpublished observations). We report here, however, that the requirements for M26 action are complex: when the M26 site with up to 3.0 kilobases (kb) of surrounding DNA was moved to other locations in the genome, it failed to enhance recombination. These results imply that the action of M26 requires another chromosomal site or proper surrounding chromatin structure. This complexity urges caution in the interpretation of large deletions that appear to locate other chromosomal sites, including recombinational hot spots.

High-Resolution Global Analysis of the Influences of Bas1 and Ino4 Transcription Factors on Meiotic DNA Break Distributions in Saccharomyces cerevisiae

“…However, the situation has been less clear for TFs in yeasts. Even those sequence motifs and their binding factors that are clearly capable of specifying strong DSB hotspot activity in some genomic contexts work poorly or not at all in other contexts (Ponticelli and Smith 1992;Mieczkowski et al 2006;this work). Moreover, presence of a sequence motif with DSB-targeting potential is by itself a poor predictor of local DSB frequency (Mieczkowski et al 2006;Fowler et al 2014).…”

Important Characteristics of Sequence-Specific Recombination Hotspots in Schizosaccharomyces pombe

“…The M26 motif generates a hotspot at many, but not all, sites in the S. pombe genome. This indicates that local DNA sequence alone is not sufficient for generating hotspots (Ponticelli and Smith 1992;Steiner and Smith 2005), which has also been demonstrated in S. cerevisiae; for example the insertion of hotspot DNA into different chromosomal contexts does not always create a new hotspot (Haring et al 2003).…”

Optimizing the Nucleotide Sequence of a Meiotic Recombination Hotspot in Schizosaccharomyces pombe

“…Thus, we tested the wt-CRE seposition within the ade6 gene (overlapping at 4 of 7 bp), it seemed unlikely that the difference in activity quence and both the active and the inactive orientations of M26 for their ability to bind Atf1-Pcr1 in vitro by gel could be due to an effect of local chromatin structure as had been previously observed with transplacements mobility shift assays. Although all of these sequences bound Atf1-Pcr1, the active allele, ade6-3086, showed of the entire ade6-M26 allele (Ponticelli and Smith 1992;Virgin et al 1995). A priori, these data imply that greater binding than either of the two inactive alleles (Figure 3), supporting the hypothesis that nucleotides hotspot activity is dependent on the orientation of the M26 heptamer at site 2, in contrast to the previously adjacent to the M26 (or CRE) sequence influence binding of the transcription factor.…”

“…Prior to the first division, homolotwo classes of hotspots have been recognized (Petes gous chromosomes recombine with each other at a 2001). ␣-Hotspots require the binding of transcription greatly elevated frequency compared to that during mifactors, whereas ␤-hotspots do not require the binding tosis (Esposito and Klapholtz 1981). This recombinaof known transcription factors.…”