MCM8 is required for a pathway of meiotic double-strand break repair independent of DMC1 in Arabidopsis thaliana

Mini-chromosome maintenance (MCM) 2-9 proteins are related helicases. The first six, MCM2-7, are essential for DNA replication in all eukaryotes. In contrast, MCM8 is not always conserved in eukaryotes but is present in Arabidopsis thaliana. MCM8 is required for 95% of meiotic crossovers (COs) in Drosophila and is essential for meiosis completion in mouse, prompting us to study this gene in Arabidopsis meiosis. Three allelic Atmcm8 mutants showed a limited level of chromosome fragmentation at meiosis. This defect was dependent on programmed meiotic double-strand break (DSB) formation, revealing a role for AtMCM8 in meiotic DSB repair. In contrast, CO formation was not affected, as shown both genetically and cytologically. The Atmcm8 DSB repair defect was greatly amplified in the absence of the DMC1 recombinase or in mutants affected in DMC1 dynamics (sds, asy1). The Atmcm8 fragmentation defect was also amplified in plants heterozygous for a mutation in either recombinase, DMC1 or RAD51. Finally, in the context of absence of homologous chromosomes (i.e. haploid), mutation of AtMCM8 also provoked a low level of chromosome fragmentation. This fragmentation was amplified by the absence of DMC1 showing that both MCM8 and DMC1 can promote repair on the sister chromatid in Arabidopsis haploids. Altogether, this establishes a role for AtMCM8 in meiotic DSB repair, in parallel to DMC1. We propose that MCM8 is involved with RAD51 in a backup pathway that repairs meiotic DSB without giving CO when the major pathway, which relies on DMC1, fails

The HEM Lines: a new library of homozygous Arabidopsis thaliana EMS mutants and its potential to detect meiotic phenotypes

Genetic screens have been crucial for deciphering many important biological processes, including meiosis. In Arabidopsis thaliana, previous forward screens have likely identified almost all the meiotic genes that when mutated lead to a pronounced decrease in fertility. However, the increasing number of genes identified in reverse genetics studies that play crucial roles in meiosis, but do not exhibit strong phenotypes when mutated, suggests that there are still many genes with meiotic function waiting to be discovered. In this study, we produced 897 A. thaliana homozygous mutant lines using Ethyl Methyl Sulfonate (EMS) mutagenesis followed by either single seed descent or haploid doubling. Whole genome sequencing of a subset of lines showed an average of 696 homozygous mutations per line, 195 of which (28%) modify a protein sequence. To test the power of this library, we carried out a forward screen looking for meiotic defects by observing chromosomes at metaphase I of male meiosis. Among the 649 lines analyzed, we identified 43 lines with meiotic defects. Of these, 21 lines had an obvious candidate causal mutation, namely a STOP or splicing site mutation in a gene previously shown to play a role in meiosis (ATM, MLH3, MLH1, MER3, HEI10, FLIP, ASY4, FLIP, PRD2, REC8, FANCL, and PSS1). Interestingly, this was the first time that six of these genes were identified in a forward screen in Arabidopsis (MLH3, MLH1, SGO1, PSS1, FANCL, and ASY4). These results illustrate the potential of this mutant population for screening for any qualitative or quantitative phenotype. Thus, this new mutant library is a powerful tool for functional genomics in A. thaliana. The HEM (Homozygote EMS Mutants) lines are available at the Versailles Arabidopsis stock center

Genetic distances and interference in <i>Atmcm8</i> using FTLs.

<p>Values are means ± Standard Error. Number of tetrads: Wild type I5aI5b n = 1986, <i>Atmcm8</i> I5aI5b n = 1022, wild type I5cI5d n = 1860, <i>Atmcm8</i> I5cI5d n = 646.</p>*<p>Z-test between wild type and <i>Atmcm8</i>.</p>**<p>The interference ratio is defined as the ratio of genetic distance of I5a with a CO in I5b by the genetic distance of I5a without a CO in I5b. The same was done for the interference ratio between I5c and I5d. Absence of interference would give a ratio of 1 that would tend to 0 with increased interference <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003165#pgen.1003165-Malkova1" target="_blank">[57]</a>. The chi square test shows a deviation from 1, and thus the presence of interference <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003165#pgen.1003165-Berchowitz1" target="_blank">[31]</a>.</p

<i>AtMCM8</i> gene structure.

<p>Exons are represented as black boxes and T-DNA insertions in <i>Atmcm8-1</i>, <i>Atmcm8-2</i> and <i>Atmcm8-3</i> alleles are indicated by triangles.</p

Epistasis tests between <i>Atmcm8</i> and <i>Atrad51</i>.

<p>Meiotic spreads with (A–B) <i>Atrad51^−/−</i>, (C–D) <i>Atmcm8^−/−/Atrad51^−/−</i>, (E–F) <i>Atrad51^+/−</i>, (G–H) <i>Atmcm8^−/−/Atrad51^+/−</i>, using DAPI staining at anaphase I and anaphase II. Bar, 10 µm.</p

Epistasis tests between <i>Atmcm8</i> and <i>sds</i> or <i>asy1</i>.

<p>Meiotic spreads with (A–B) <i>sds^−/−</i>, (C–D) <i>Atmcm8^−/−/sds^−/−,</i> (E–F) <i>asy1^−/−</i>, (G–H) <i>Atmcm8^−/−/asy1^−/</i>, using DAPI staining at anaphase I and anaphase II. Bar, 10 µm.</p

Coimmunolocalization of ASY1 and ZYP1.

<p>ASY1 (red), ZYP1 (green) are shown as well as the overlay of both signals (merge) at pachytene in (A) wild type and in (B) <i>Atmcm8</i> mutant. In both wild type and mutant the polymerization of the synaptonemal complex, revealed by ZYP1, is completed at pachytene. The ASY1 signal is largely depleted from the chromosomes as the synaptonemal complex forms. Bar, 10 µm.</p

Alexander staining.

<p>(A) A wild type anther containing pollen grains that are all viable, as indicated by their red staining and round shape. (B) An <i>Atmcm8-1</i> anther containing viable and dead pollen grains as indicated by their abnormal shapes and green coloration. Bar, 100 µm.</p

Seed per fruit and fragmentation levels in different combinations of double mutants.

<p>Values are means ± Standard Error. The number of fruit or cells counted is indicated in brackets. ND: not determined, a–e: indicates significant differences among groups (Newman Keuls test, p>0.05). Number of crosses indicates fragmentation levels, based on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003165#pgen-1003165-g005" target="_blank">Figure 5</a>.</p

Epistasis tests between <i>Atmcm8</i> and mutants affected in crossover formation.

<p>Meiotic spreads with (A–C) <i>Atmsh4</i>, (D–F) <i>Atmcm8/Atmsh4</i>, (G–I) <i>Atmus81</i>, (J–L) <i>Atmcm8/Atmus81</i>, (M–O) <i>Atmsh4/Atmus81</i>, (P–R) <i>Atmcm8/Atmsh4/Atmus81</i> using DAPI staining at metaphase I, anaphase I and anaphase II. Bar, 10 µm.</p