Replication-dependent early meiotic requirement for Spo11 and Rad50

Abstract

Spo11 and the Rad50-Mre11 complex have been indirectly implicated in processes associated with DNA replication. These proteins also have been shown to have early meiotic roles essential for the formation of a programmed DNA double-strand break known in Saccharomyces cerevisiae to initiate meiotic recombination. In both S. cerevisiae and the basidiomycete Coprinus cinereus, spo11 and rad50 mutants are defective in chromosome synapsis during meiosis. Here we demonstrate that a partial restoration of synapsis occurs in C. cinereus spo11 and rad50 mutants if premeiotic DNA replication is prevented. Double mutants were constructed with spo11-1 or rad50-4 and another mutant, spo22-1, which does not undergo premeiotic DNA replication. In both cases, we observed an increase in the percentage of nuclei containing synaptonemal complex (SC) structures, with concomitant decreases in the percentage of nuclei containing axial elements (AE) only or no structures. Both types of double mutants demonstrated significant increases in the average numbers of AE and SC, although SC-containing nuclei did not on average contain more AE than did nuclei showing no synapsis. Our results show that Spo11-induced recombination is not absolutely required for synapsis in C. cinereus, and that the early meiotic role of both Spo11 and Rad50 in SC formation partially depends on premeiotic S phase. This dependency likely reflects either a requirement for these proteins imposed by the premeiotic replication process itself or a requirement for these proteins in synapsis when a sister chromatid (the outcome of DNA replication) is present.

Figure 1

SC formation in wild-type and mutant strains. (A) TSM1, wild type; (B) TSM3, spo11–1; (C) TSM4, rad50–4; (D) TSM2, spo22–1; (E) TSM5, spo11–1;spo22–1; and (F) TSM8, rad50–4;spo22–1. [Scale bars = 2 μm.]

Figure 2

Comparison of wild-type, single-, and double-mutant strains. Chromosome spreads were examined and categorized according to the presence or absence of AE and SC. For each strain, at least two mushrooms were sampled. Total numbers of spreads were: TSM1, 22; TSM2, 19; TSM3, 60; TSM4, 30; TSM5, 27; TSM6, 45; TSM7, 38; TSM8, 40; and TSM9, 27.

Figure 3

Comparison of SC and AE length vs. the number of SC and AE segments. Total SC and AE lengths were measured and the sum was divided by the total number of nuclei to determine the average length per nucleus for each strain listed in the table. For each chromosome spread, AE segments were counted as one segment for each continuous piece regardless of length. A single SC segment represents continuous association of two AE. Data are presented as ± indicates the SEM. SC and AE lengths are in micrometers (μm).