Abstract:
Crossover recombination (COs) is central to meiosis where reciprocal exchange of chromosome fragments between parental genomes occurs, shuffling chromosomes and making each offspring unique. COs are formed via homologous repair of programmed DNA double-strand breaks (DSBs) by two major pathways – the Class I pathway, catalysed by a meiotic specific group of proteins known as ZMMs, represent most COs; the minor Class II pathway forms the remaining COs, also involved in DSB repair of somatic cells. Despite DSBs occurring throughout the genome, CO placement is precisely patterned and subject to a phenomenon termed interference where COs on the same chromosome do not occur in close proximity, the mechanism of which remains elusive. To identify novel actors that regulate CO designation along chromosomes during meiosis, we adopt TurboID(TbID) based proximity labelling for meiotic cells of Arabidopsis thaliana. TbID tagged transgenic lines to the meiotic chromosome axis proteins REC8, ASY1/3; the transverse element of the synaptonemal complex ZYP1a/b; ZMM proteins ZIP4, HEI10 and the MutLγ resolvase MLH3 were employed as baits. Interestingly examining complementation of these transgenic lines revealed tagging REC8 by the TbID construct results in fertility loss supposedly due to premature depletion in pericentromeric REC8, serving as valuable tool to study centromere proximal COs in the future. Current evidence suggests that the ZMM protein HEI10, which encodes a conserved RING domain containing E3 ubiquitin ligase plays a more central role in CO designation through a diffusion- mediated coarsening process. Here, we discover a RING domain mutant of AtHEI10 showing a significant decrease in fertility, highlighting the indispensable role of this domain in CO patterning plausibly through ubiquitination. Further, by mutating the Leucine 74 residue of HEI10 predicted to impede HEI10 tetramerisation based on Alfafold2 predictions, we also show that higher-order interactions of HEI10 might plausibly govern its coarsening dynamics. Taken together this study further refines our understanding of CO patterning during meiosis.
