The control of Spo11's interaction with meiotic recombination hotspots

Abstract

Programmed double-strand breaks (DSBs), which initiate meiotic recombination, arise through the activity of the evolutionary conserved topoisomerase homolog Spo11. Spo11 is believed to catalyze the DNA cleavage reaction in the initial step of DSB formation, while at least a further 11 factors assist in Saccharomyces cerevisiae. Using chromatin-immunoprecipitation (ChIP), we detected the transient, noncovalent association of Spo11 with meiotic hotspots in wild-type cells. The establishment of this association requires Rec102, Rec104, and Rec114, while the timely removal of Spo11 from chromatin depends on several factors, including Mei4 and Ndt80. In addition, at least one further component, namely, Red1, is responsible for locally restricting Spo11's interaction to the core region of the hotspot. In chromosome spreads, we observed meiosis-specific Spo11-Myc foci, independent of DSB formation, from leptotene until pachytene. In both rad50S and com1⌬/sae2⌬ mutants, we observed a novel reaction intermediate between Spo11 and hotspots, which leads to the detection of full-length hotspot DNA by ChIP in the absence of artificial cross-linking. Although this DNA does not contain a break, its recovery requires Spo11's catalytic residue Y135. We propose that detection of uncross-linked full-length hotspot DNA is only possible during the reversible stage of the Spo11 cleavage reaction, in which rad50S and com1⌬/sae2⌬ mutants transiently arrest.[Keywords: spo11-Y135; cleavage complex; "tight" binding; DSB repair; Spo11 cleavage model] Supplemental material is available at http://www.genesdev.org.

Interactions between Mei4, Rec114, and other proteins required for meiotic DNA double-strand break formation in Saccharomyces cerevisiae

“…The findings in this study reinforce the view that has emerged from several lines of inquiry that there are at least four functionally distinct subgroups among the DSB proteins: Spo11-Ski8; Rec102-Rec104; Mer2-Mei4-Rec114; and Mre11-Rad50-Xrs2 (see Introduction) (Ohta et al 1998;Jiao et al 2003;Arora et al 2004;Kee et al 2004;Prieler et al 2005;Henderson et al 2006;Li et al 2006;Sasanuma et al 2007). To date, there is little support for the possibility that all of the DSB proteins together form a discrete "DSB holoenzyme," although available data cannot rule out this possibility.…”

“…Moreover, Mer2 and Rec114 each localize to chromosomes independently of the other members of the group, and indeed Mer2 associates with chromosomes even in vegetative cells (this study and Henderson et al 2006;Li et al 2006). Finally, Rec114 but not Mei4 or Mer2 is required for association of Spo11 with DSB hotspot sequences as assessed by chromatin immunoprecipitation (Prieler et al 2005) and for the ability of Gal4BD-Spo11 to recruit other Spo11 molecules to a Gal4 binding site on chromatin (Sasanuma et al 2007). The available data thus reveal a complex set of partially dependent and partially independent relationships between these proteins.…”

“…Among the meiotically induced DSB proteins, Rec102 and Rec104 share perhaps the most intimate relationship with one another: they interact physically and genetically, they are mutually interdependent for proper chromosome localization, their behaviors are affected (or not) in parallel ways by mutations in other genes, and absence of either one exerts the same kind of effect (or not) on the behavior of other proteins (Salem et al 1999;Jiao et al 2003;Arora et al 2004;Kee et al 2004;Prieler et al 2005;Henderson et al 2006;Li et al 2006;Sasanuma et al 2007). The yeast two-hybrid results presented here further support this conclusion.…”

“…Physical and functional connections of Rec102-Rec104 with Spo11 are well established (Salem et al 1999;Kee and Keeney 2002;Jiao et al 2003;Arora et al 2004;Kee et al 2004;Prieler et al 2005;Sasanuma et al 2007). In contrast, it has been less clear how Mei4 and Rec114 are connected to Spo11, and thus how they are able to influence DSB formation.…”

“…Although this Rec102-Rec104 complex interacts with other DSB proteins, including Spo11, there are numerous differences between Rec102-Rec104 and the other proteins with respect to their genetic dependencies for protein-protein and protein-chromosome interactions Wells et al 2006) (discussed in more detail below). Moreover, whereas Mre11 and Spo11 appear to localize specifically to preferred sites of DSB formation as assessed by chromatin immunoprecipitation, Rec102 appears to be more broadly associated with both hotspot and non-hotspot regions (Borde et al 2004;Prieler et al 2005). …”