Bas1p binding on yeast chromosome XI in cells incubated for 3 hours in sporulation medium (A) or grown in rich medium (YPD) (B). We performed chromatin immunoprecipitation using an epitope-tagged Bas1p and used Bas1p-associated DNA as a hybridization probe on a microarray. The resulting data were analyzed using a 1,000-bp window that was moved at intervals of 250 bp (ChIPOTle version 1.0). Bas1p binding is considerably stronger in cells incubated in sporulation medium (A) than in cells grown in rich medium (B). Neither KIT12 nor SRP40 has a consensus Bas1p binding site in the promoter region, although KIT12 has a perfect match to the consensus within its coding sequence.

Consensus Bas1p binding site and location of the binding site relative to the initiation codon of the target gene. (A) We used the MDscan program (38) and identified a sequence associated with Bas1p binding; the core motif is the same as that identified by Daignan-Fornier and Fink (14) as a Bas1p binding site. (B) We determined the location of the Bas1p binding motif in upstream regions identified as bound (red) and unbound (blue) by the microarray analysis. The distances shown on the x axis are given relative to the initiating codon of the target ORF.

Relationship between Bas1p binding and metabolic pathways concerned with histidine and adenine biosynthesis and one-carbon metabolism. This diagram is an extension of Fig.7 of reference 21. Genes identified in our study as binding Bas1p in the promoter regions are shown in red. The abbreviations in the adenine biosynthetic pathway are as follows: PRPP, 5-phosphoribosyl diphosphate; PRA, 5-phosphorybosylamine; GAR, 5′-phosphoribosylglycinamide; FGAR, 5′-phosphoribosyl-N′-formylglycinamide; FGAM, 5′-phosphoribosyl-N′-formylglycinamide; AIR, 1-(5′-phosphoribosyl)-5-aminoimidazole; CAIR, 1-(5′-phosphoribosyl)-5-aminoimidazole-4-carboxylate; SAICAR, 1-(5′-phosphoribosyl)-4-(N-succinocarboxyamido)-5-aminoimidasole; AICAR, 5-amino-1-(5′-phosphoribosyl)-imidazole-4-carboxyamide; FAICAR, 5-formamido-1-(5′-phosphoribosyl)-imidazole 4-carboxyamide; THF, tetrahydrofolate.

Recombination activities of all ORFs in the wild-type strain JG169. We used GeneSpring software to show the relative recombination activities. The color code on the right indicates the median ratio of hybridization (DSB-enriched sample/total genomic DNA), with red representing hot spots and blue representing cold spots. No data were recorded for ORFs color coded as gray. The black ovals represent the centromeres, and the red arrow above chromosome XII indicates the position of the rRNA gene cluster; on the microarray, only 2 of the 100 repeats are represented. Thus, the suppression of recombination extends about 100 kb to each side of the cluster.

Comparison of recombination activities on Bas1p targets on chromosome III in wild-type and bas1 strains. As shown in panels A (wild-type strain) and B (bas1 strain), the recombination activities of most regions are unaffected by the bas1 mutation. One exception is the HIS4 gene, where the recombination activity is significantly reduced, as expected from our previous study (56). In panel C, we show Bas1p binding (in meiotic cells) with a single chromosome peak at the position of the HIS4 gene.

Context-dependent effects of the bas1 deletion on local meiosis-specific DSBs of four genes (HIS4, SHM2, ADE8, and GCV2) that have upstream Bas1p binding sites and effects of deletion of Bas1p binding sites upstream of SHM2 on local meiosis-specific DSB formation. (A) Recombination activities of small chromosome regions containing the target gene, expressed as ranked values. Activities in the wild-type and bas1 strains are represented with blue and red lines, respectively. The recombination activities of the HIS4 and SHM2 genes are significantly reduced by the bas1 mutation, recombination of the ADE8 gene is unaffected, and the recombination activity of the MRSP17-GCV2 genes is increased. (B) Southern analysis confirming the microarray data for the same four genes. The positions of the meiosis-specific DSBs are indicated by arrows. The restriction enzymes used for the analysis of HIS4, SHM2, ADE8, and GCV2, respectively, were BglII, NcoI, EcoRV, and StuI. wt, wild type. (C) Southern analysis of a strain (MD341) in which the two Bas1p binding sites upstream of SHM2 were altered (TGACTC to AAACTC). The strain homozygous for the resulting mutation (SHM2-1) lacked the meiosis-specific DSBs (indicated by arrows) upstream of SHM2 that were present in the wild-type strain (EcoRI digest of genomic DNA). The filters were stripped and rehybridized to a probe derived from YGR177C (described in the supplemental material), a gene associated with a very strong DSB. Similar levels of DSBs were observed in both samples (data not shown).