Targeted induction of meiotic double-strand breaks reveals chromosomal domain-dependent regulation of Spo11 and interactions among potential sites of meiotic recombination.
Meiotic recombination is initiated by programmed DNA double-strand break (DSB) formation mediated by Spo11. DSBs occur with frequency in chromosomal regions called hot domains but are seldom seen in cold domains. To obtain insights into the determinants of the distribution of meiotic DSBs, we examined the effects of inducing targeted DSBs during yeast meiosis using a UAS-directed form of Spo11 (Gal4BD-Spo11) and a meiosis-specific endonuclease, VDE (PI-SceI). Gal4BD-Spo11 cleaved its target sequence (UAS) integrated in hot domains but rarely in cold domains. However, Gal4BD-Spo11 did bind to UAS and VDE efficiently cleaved its recognition sequence in either context, suggesting that a cold domain is not a region of inaccessible or uncleavable chromosome structure. Importantly, self-association of Spo11 occurred at UAS in a hot domain but not in a cold domain, raising the possibility that Spo11 remains in an inactive intermediate state in cold domains. Integration of UAS adjacent to known DSB hotspots allowed us to detect competitive interactions among hotspots for activation. Moreover, the presence of VDE-introduced DSB repressed proximal hotspot activity, implicating DSBs themselves in interactions among hotspots. Thus, potential sites for Spo11-mediated DSB are subject to domain-specific and local competitive regulations during and after DSB formation.
Fukuda
Tomoyuki
T
Shibata Distinguished Senior Scientist Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. tfukuda@riken.jp
Kugou
Kazuto
K
Sasanuma
Hiroyuki
H
Shibata
Takehiko
T
Ohta
Kunihiro
K
D001665:Binding Sites; D015825:Chromosomes, Fungal / Q000737:chemistry*; D053903:DNA Breaks, Double-Stranded; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases; D008540:Meiosis / Q000235:genetics*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D000099257:Meiotic Recombination Protein SPO11
D004250:DNA Topoisomerases, Type II; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases; D006180:Proton-Translocating ATPases; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C052130:GAL4 protein, S cerevisiae; C518938:Spo11 protein, S cerevisiae; D000099257:Meiotic Recombination Protein SPO11; C081687:VMA1 protein, S cerevisiae