Functional organization of protein determinants of meiotic DNA break hotspots

Lijuan Ma,Kyle R Fowler,Cristina Martín-Castellanos,Gerald R Smith

doi:10.1038/s41598-017-00742-3

Abstract

During Schizosaccharomyces pombe meiotic prophase, homologous chromosomes are co-aligned by linear elements (LinEs) analogous to the axial elements of the synaptonemal complex (SC) in other organisms. LinE proteins also promote the formation of meiotic DNA double-strand breaks (DSBs), the precursors of cross-overs. Rec10 is required for essentially all DSBs and recombination, and three others (Rec25, Rec27, and Mug20) are protein determinants of DSB hotspots – they bind DSB hotspots with high specificity and are required for DSB formation there. These four LinE proteins co-localize in the nucleus in an interdependent way, suggesting they form a complex. We used random mutagenesis to uncover recombination-deficient missense mutants with novel properties. Some missense mutations changed essential residues conserved among Schizosaccharomyces species. DSB formation, gene conversion, and crossing-over were coordinately reduced in the mutants tested. Based on our mutant analysis, we revised the rec27 open reading frame: the new start codon is in the previously annotated first intron. Genetic and fluorescence-microscopy assays indicated that the Rec10 N- and C-terminal regions have complex interactions with Rec25. These mutants are a valuable resource to elucidate further how LinE proteins and the related SCs of other species regulate meiotic DSB formation to form crossovers crucial for meiosis.

Highlights

Meiosis is a special process in which diploid cells undergo one round of DNA replication followed by two consecutive rounds of chromosome segregation to generate haploid gametes
In meiotic prophase of the fission yeast Schizosaccharomyces pombe, linear elements (LinEs), but not full-length synaptonemal complex (SC), are detected by electron microscopy (EM) of spread-out nuclear contents; LinEs appear similar to lateral elements of the SC of other species but do not extend end-to-end on the chromosomes in such EM analyses[3, 4]
Spores were spotted on YEA plates without supplementary adenine, on which non-recombinant (Ade−) spores form red colonies, while recombinant (Ade+) spores form white colonies; this produces a red lawn with some white (Ade+) papillae

Summary

Introduction

Meiosis is a special process in which diploid cells undergo one round of DNA replication followed by two consecutive rounds of chromosome segregation to generate haploid gametes. Homologous recombination is initiated by DNA double-strand breaks (DSBs) formed by the conserved protein Spo[11] (called Rec[12] in fission yeast, studied here)[1]. Rec[8] forms filamentous structures with the chromosomes aligned in parallel from one end to the other during the horsetail stage of meiosis as observed in live cells by super-resolution structured illumination microscopy (SIM)[7] This observation reveals a structural similarity of S. pombe meiotic chromosomes and those of other species. In the absence of Mug[20], LinEs fail to develop to full-length[12] or to form distinct nuclear foci[16] These four LinE components interdependently co-localize in meiotic nuclei of live cells; i.e., focus-formation of any one depends on each of the others tested[11, 16]. Hop[1] and Mek[1], localize to LinEs and their localization depends on Rec[109]

Methods

Results

Conclusion