Crossover assurance and crossover interference are distinctly regulated by the ZMM proteins during yeast meiosis Miki Shinohara 1 , Steve D Oh 2 , Neil Hunter 2 & Akira Shinohara 1,3 Meiotic crossing-over is highly regulated such that each homolog pair typically receives at least one crossover (assurance) and adjacent crossovers are widely spaced (interference). Here we provide evidence that interference and assurance are mechanistically distinct processes that are separated by mutations in a new ZMM (Zip, Msh, Mer) protein from Saccharomyces cerevisiae, Spo16. Like other zmm mutants, spo16 cells have defects in both crossing-over and synaptonemal complex formation. Unlike in previously characterized zmm mutants, the residual crossovers in spo16 cells show interference comparable to that in the wild type. Spo16 interacts with a second ZMM protein, Spo22 (also known as Zip4), and spo22 mutants also show normal interference. Notably, assembly of the MutS homologs Msh4 and Msh5 on chromosomes occurs in both spo16 and spo22, but not in other zmm mutants. We suggest that crossover interference requires the normal assembly of recombination complexes containing Msh4 and Msh5 but does not require Spo16- and Spo22-dependent extension of synaptonemal complexes. In contrast, crossover assurance requires all ZMM proteins and full-length synaptonemal complexes. Crossing-over during meiosis establishes the physical linkage of homologs required for their accurate segregation at the first meiotic division (see ref. 1 for review). Two features indicate that meiotic crossing-over is a highly regulated process 1,2 . First, ‘crossover assur- ance’ or ‘the obligatory crossover’ describes the observation that each pair of homologs typically obtains at least one crossover despite the fact that the average number of crossovers per chromosome pair is very low (typically one to three). Second, adjacent crossovers are more widely spaced than random expectations would suggest, a phenom- enon known as crossover interference 3 . Meiotic recombination is initiated by DNA double-strand breaks (DSBs) 4 , and the total number of DSBs greatly exceeds the final number of crossovers. Thus, there must be processes that designate a crossover fate for selected DSBs and then implement that fate with high efficiency. The remaining majority of DSBs are repaired as noncrossovers without exchange of chromosome arms. At the DNA level, differentiation of the crossover and noncrossover pathways can be detected at an early stage. DSBs undergo resection of the 5¢ strands to yield long, 3¢, single-stranded tails. DSB ends then interact sequentially with a homolog to form two types of joint molecule intermediates. Invasion by one DSB end produces a ‘single-end invasion’ (SEI) 5 . DNA synthesis and interaction with the second end then convert the SEI into a double Holliday junction (dHJ) 6 . In theory, dHJs can be resolved into both crossover and noncrossover products 7 , but available evidence suggests that dHJs give rise primarily or exclusively to crossovers 5,8 . Moreover, SEIs also seem to be crossover-specific intermediates 5,9 . Joint molecules that are specific to the noncrossover pathway have not been identified, perhaps because they are labile and/or short-lived. It is proposed that the crossover or noncrossover designation occurs very early—at or before the transition from DSBs to SEIs 1,5,9 . Meiotic recombination also mediates homolog pairing, culminating in the formation of synaptonemal complexes, which connect homo- logs along their entire lengths 10 . Meiotic homologs assemble a proteinaceous core or axis. During the leptotene stage, DSBs form and homolog axes pair, becoming closely associated at sites of recombination 11 . These axial associations are thought to be sites where synaptonemal complex polymerization initiates during the zygotene stage. When synaptonemal complexes have polymerized along the lengths of all homolog pairs, cells enter the pachytene stage, and dHJs are formed and resolved into crossovers 11 . The meiosis-specific ZMM proteins coordinate recombina- tion and synaptonemal complex formation in Saccharomyces cerevisiae. Members of the ZMM group represent a diverse collection of proteins: Zip1 is the major central element compo- nent of synaptonemal complexes; Zip2 is related to WD-repeat proteins; Zip3 is a SUMO E3 ligase; Mer3 is a 5¢–3¢ DNA helicase; the MutS homologs Msh4 and Msh5 form a complex that can bind Holliday junctions; and Spo22 (also known as Zip4) is a TPR-like repeat protein 12–21 . zmm mutants Received 6 July 2007; accepted 11 December 2007; published online 24 February 2008; doi:10.1038/ng.83 1 Institute for Protein Research, Osaka University, Suita 565-0871, Japan. 2 Sections of Microbiology and Molecular & Cellular Biology, University of California Davis, Davis, California 95616, USA. 3 Graduate School of Science, Osaka University, Suita 565-0871, Japan. Correspondence should be addressed to A.S. (ashino@protein.osaka-u.ac.jp). NATURE GENETICS VOLUME 40 [ NUMBER 3 [ MARCH 2008 299 ARTICLES © 2008 Nature Publishing Group http://www.nature.com/naturegenetics