The Basic Chromosome Pairing and Recombination Process

In many species, there are chromosome movements at the onset of meiosis, which enable the telomeres of the chromosomes to cluster as a telomere bouquet. Within this cluster of telomeres, the terminal regions of homologous chromosomes find their correct homologous partner. The two homologues then “zip up” from the telomere regions. This process of “zipping up” or synapsis involves the placement of a protein complex, a synaptonemal complex, between the homologues. Essentially this is equivalent to “gluing” the chromosomes together. Within this synaptonemal complex structure, double strand breaks can be repaired. Meiotic recombination occurs through the generation and repair of double strand breaks (DSBs) using the homologues or homoeologues. Essentially the double strand break is formed early during meiosis and is then resectioned to generate a single strand end. The pairing process involves the single strand end finding and invading the homologous region of the corresponding homologue or homoeologue. Successful invasion results in strand displacement in this region, generation of a Double Holiday Junction, a cross-over event (chiasmata) leading to recombination. The single strand invasion occurs during late zygotene, and double holiday formation occurs during pachytene. The synaptonemal complex (SC) starts to be disassembled during pachytene. The formation of chiasmata, physical links which together with sister chromatid cohesion, still hold the homologues together after the disassembly of the SC, so the chromosomes are visualized as paired at metaphase I. If single strand invasion is unsuccessful, the double strand break can be repaired through using its own sister chromatid, leading to the chromosomes being visualized as unpaired at metaphase I.

The Power of a Cell Biological Experiment

After 50 years, the cell biological tools (antibodies to key meiotic proteins) finally became available to answer two fundamental questions about Ph1.

Firstly, the question as to whether Ph1 actually blocks chromosome pairing between related chromosomes (homoeologues). The locus is named homoeologous pairing 1 (Ph1) because it is always assumed that it reduces pairing or synapsis between homoeologues, and that this then subsequently affects the levels of recombination between such chromosomes. However, our recent cell biology data reveals that in wheat-rye hybrids, where there are no homologues, only homoeologues, the related chromosomes pair or synapse to a similar level, whether Ph1 is present or absent (Martin et al. 2014). Therefore Ph1 doesn't suppress homoeologues pairing in the hybrid. This implies that in wheat itself where there are both homologues and homoeologues, the overall effect of Ph1 on chromosome pairing as distinct from recombination must be the promotion of homologue pairing rather than specifically suppressing pairing between the related chromosomes.

Secondly at what stage does Ph1 block recombination between homoeologues, a process that occurs on both the male and female sides from leptotene to diplotene? The major surprise of our recent study is that double strand breaks are formed at similar levels, and are processed with similar kinetics into Double Holliday Junctions between the paired homoeologues whether Ph1 is present or absent (Martin et al. 2014). This results in a similar number of Double Holliday Junctions at diplotene in the hybrid as revealed by immunolabelling with the MLH1 antibody. In all other species so far studied, MLH1 marks sites on paired chromosomes that will become crossovers. However, these studies have been performed on paired homologues rather than paired homoeologues as in the case of the wheat-rye hybrid. Based on the number of MLH1 sites, 21 crossovers would be expected between the paired homoeologues in the wheat-rye hybrid whether Ph1 is present or absent, yet only seven crossovers on average occur in the absence of Ph1, and one or none in the presence of Ph1. Therefore the resolution of Double Holliday Junctions to crossovers between the paired homoeologues fails in both the presence and absence of Ph1, but this failure is partially alleviated by deleting Ph1 or increasing Cdk2type activity. Thus Ph1 suppresses recombination between homoeologues by preventing the resolution of Double Holliday Junctions as crossovers (Martin et al. 2014).

The MLH1 protein complex involved in this resolution has been characterized in other species. It contains two mismatch repair proteins, MLH1 and MLH3, EXO1 (a nuclease), CDK2 (which is activated by a meiotic specific cyclin), and finally an E3 Ubiquitin ligase (HEI10), which may be involved in the degradation of other cyclins, enabling the meiotic cyclin to activate CDK2. Various recent reports have provided indirect evidence that CDK2 regulates the activity of the MLH1 complex and crossovers (Martin et al. 2014).

Thus this cell biological study reveals that Ph1 has two distinct effects on chromosome pairing and recombination. Firstly it promotes homologue pairing rather than prevent homoeologue pairing, and secondly it prevents recombination between paired homoeologues by stalling Double Holliday Junctions from being resolved as crossovers (Martin et al 2014). Interestingly subsequently a number of Cdk2 studies have reported that it has two distinct effects on chromosome pairing and recombination. It affects chromosome pairing through altering the function of the telomere bouquet, and recombination via crossover resolution (Liu et al. 2014; Viera et al. 2015). So what is Ph1 locus?

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