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

Abstract

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.

Figure 1.—

Method for screening a large number of unique 15- or 30-bp sequences for hotspot activity. (A) A strain (WS129) with a partial ade6 deletion and ura4⁺-kanMX6 insertion and plasmid pWS35 (not shown) is transformed with linear ade6 DNA containing a 15- or 30-bp random sequence substitution (shaded bar). (B) Homologous recombinants lose the ura4⁺-kanMX6 marker and now share homology to a fragment of ade6 carried on the plasmid pWS35. After transfer to sporulation medium, these cells will self-mate and form spores. Strains containing a hotspot in the random sequence region will recombine with the plasmid at high frequency (open arrow) to form ade6⁺ spores, which can be identified as white papillae on red colonies after replica plating to the appropriate medium (Figure 2). Although this figure implies noncrossover conversion to ade6⁺, crossover recombinants would also likely produce ade6⁺ spores, because the plasmid carries 164 bp of the ade6 promoter, which is adequate for ade6 expression (Zahn-Zabal et al. 1995). ade6 genes and fragments are drawn to scale; other genes and constructs are not. Open rectangles represent open reading frames.

Figure 2.—

Visual assay for hotspots. Strains containing known hotspot alleles (ade6-M26 or -3074) and one control allele (ade6-M375) were put through the steps of the screen described in the text. (A) ade6-M375, (B) ade6-M26, (C) ade6-3074, (D) ade6-M375 (left), and ade6-3074 (right). A larger version of this figure is shown in Figure S2.

Figure 3.—

Reconstructed sequence motifs produce hotspots. High-frequency sequence motifs (Table 2 and Figure S1) were reconstructed in the ade6 gene and tested for hotspot activity in homothallic crosses (plasmid pWS35 × chromosome; solid bars) or heterothallic crosses (chromosome × chromosome; open bars). Heterothallic strains were crossed with WS315 (ade6-469; Table 1). Each bar represents the average of at least three crosses ±1 SEM. Sequences show the original motif (Table 2; boldface type) with some flanking nucleotides (regular type). Consensus sequence nucleotides (Figure S1) are underlined when applicable. In this table, only ade6-4009 and ade6-4094 are not significantly more active than ade6-M375 (P > 0.01, Student's t-test). Notes: ^aThe motif (Table 2) on which the allele is based. Con, consensus sequence. NA, not applicable. Motifs with three numbers indicate reconstruction of a particular allele from a subscreen (Figure S1), e.g., 7-31-16 = sequence number 16 from subscreen of motif 7-31. ^bThese motifs are inverted relative to ade6-4100 and -4104. ^cThese motifs are located 36 bp downstream relative to ade6-4099 and -4103.

Figure 4.—

The CCAAT-binding factor is required specifically for activity of the ade6-4002 hotspot, but not ade6-M26. Crosses were performed between heterothallic strains containing the indicated ade6 alleles and mutations in the php2, php3, or php5 genes, which encode subunits of the CCAAT-binding factor. Bars represent the average Ade⁺ recombinant frequencies ± SEM from a minimum of three experiments for each cross.

Figure 5.—

Potential hotspot families. Hotspot motifs (Figure 3) and hotspot consensus sequences (Figure S1) are aligned to show similarities. In the CRE family of hotspots, bases differing from the 10-bp CRE palindrome (ade6-3083, Steiner and Smith 2005b) are underlined. Comp, complement of consensus sequence (Figure S1).

Figure 6.—

Model to explain the location of recombination hotspots. The large rectangle indicates a portion of a chromosome. Shaded regions indicate regions of the genome that are permissive for recombination hotspots. In S. pombe, these regions coincide primarily with large intergenic regions (Cromie et al. 2007). Solid lines indicate hotspot sequence motifs. Hotspots occur where these two chromosomal features coincide.