Meiosis is a specialized cell division used by sexually reproducing organisms to generate cells with half the number of chromosomes present in the parent cells. These cells are called gametes and named ova and sperm in humans. In asexual cell division, called mitosis, chromosomes are copied in one round of DNA replication and then the copies are segregated between two daughter cells. When a cell undergoes meiosis, one round of DNA replication is followed by two successive rounds of chromosome segregation, generating 4 daughter cells with half the number of chromosomes present in the original cell. In the early stages of meiosis, homologous chromosome pairs associate with each other and exchange sequences. This exchange, called homologous recombination, generates genetically unique gamete cells. It also helps the chromosomes segregate properly and therefore protects against miscarriage and Down syndrome.
Image courtesy of K. Fowler and G. Smith.
In fission yeast, protein complexes called linear elements (LinE) bind along the chromosomes and aid recombination of the DNA duplexes. The LinE complex has structural and functional similarities to the synaptonemal complex (SC) responsible for intimate chromosome pairing in most eukaryotic organisms studied. Prior to recombination, a special protein called Spo11 (or Rec12 in fission yeast) generates double-strand breaks (DSBs) in the DNA. The breaks are not evenly distributed across chromosomes but instead tend to form at so-called hotspots in addition to more rarely cut (”cold”) regions. Scientists in the Smith Lab (Basic Sciences Division) study DSB formation and recombination using fission yeast as a model organism. "DSBs are potentially dangerous depending on the chromosomal site, so cells must do some decision-making on a site-by-site basis," said Mr. Fowler. How are the DSB sites chosen and broken, and how is the DNA exchanged or repaired? These questions drive ongoing investigation.
To generate new data to address these questions, scientists in the Smith Lab carried out a genetic screen and isolated 90 new mutations in LinE complex proteins that disrupt DSB formation and recombination. Their results were recently published in Scientific Reports.
In the screen, they mutagenized each of 3 essential LinE genes, rec25, rec27, and mug20, using an error-prone DNA polymerase. Next, they transformed mutated plasmids into strains lacking the endogenous copy of the mutated gene. For example, they made strains which carried a mutant rec25 plasmid and which had the endogenous rec25 genes deleted (rec25Δ/rec25Δ). In addition to lacking a functional copy of the tested LinE gene, the diploid yeast strains had two non-complementing mutations in the ade6 gene. These strains appear red when grown on media with low adenine. The mutations in the ade6 genes recruit the LinE proteins and wild-type strains will therefore recombine to produce Ade6+ haploid spores, which appear as white colonies on the low adenine media, at some frequency. Therefore, the scientists screened for the ability of the mutated version of each of the LinE genes to carry out recombination by screening for the appearance of white colonies, which can only be formed when the mutated ade6 genes recombine following DSB formation.
Through this screen, they identified 43 rec25 mutants, 32 rec27 mutants, and 15 mug20 mutants that were defective in homologous recombination and/or DSB formation. They characterized these mutants by sequencing them and also used fluorescence microscopy to visualize the formation of LinE complex foci in some of the mutants. They found that individual or pairs of mutations altering amino-acid residues in Rec25, some of which are conserved among fission yeast strains, resulted in decreased recombination that was similar to a complete deletion of the gene (rec25Δ). Thus, those residues are essential to Rec25 function. Additionally, they found that mutation of a conserved KR patch (lysines and arginines) in the N-terminus of Rec27 strongly reduced recombination.
Image courtesy of L. Ma, C. Martin-Castellanos, and G. Smith.
In addition to the screen, the scientists sequenced 7 previously isolated (but not sequenced) and 1 newly isolated mutant of Rec10, a LinE protein essential for all meiotic recombination. The new mutant, rec10-216, was created when two N-terminal codons that are conserved among fission yeast were randomly mutated. They found that rec10-216 altered the formation of Rec25 foci in cells and they hypothesized that this is due to inefficient movement of Rec25 into the nucleus or retention there (see figure). In addition, they performed genetic complementation analysis and their results suggest that the C-terminus of Rec10 interacts with Rec25.
A failure to properly recombine could result from an inability to form or repair double-strand breaks (DSBs). In order to analyze whether the formation of DSBs was compromised in the mutants, the scientists performed Southern blots probing for DNA DSBs at a known hotspot location (the ade6-3049 hotspot). Yeast with mutations in DSB repair proteins such as Rad50 accumulate breaks at this hotspot whereas rad50 mutant strains with additional mutations altering recombination proteins (Rec- mutants) do not form breaks. Indeed, the scientists found that newly isolated mutants with a strong lack of recombination did not form DNA breaks, and intermediate mutants showed reduced break formation at the site.
"DNA double-strand breaks (DSBs) and crossover connections between homologous chromosomes are critical for meiosis and making viable sex cells (eggs and sperm in humans)," said Dr. Smith. "The mutants we studied here will help understand how DSB formation is properly regulated both in position and frequency along chromosomes and thereby aids sexual reproduction.”
Ma L, Fowler KR, Martin-Castellanos C, Smith GR. 2017. "Functional organization of protein determinants of meiotic DNA break hotspots." Scientific Reports.
This research was supported by the National Institutes of Health (to GRS), the Spanish Ministry of Science and Innovation, and the Spanish Ministry of Economy and Competitiveness (to CMC).