Biology subjects

Arter, M.

Publications and source records attributed to Arter, M..

4 recordsLinked to original sources

bioRxiv2025

Structure-informed evolutionary analysis of the meiotic recombination machinery

Despite being essential for fertility, many proteins involved in meiotic homologous recombination have diverged rapidly. The evolutionary forces driving this divergence remain mostly unknown, in part because of challenges in accounting for the interplay of sequence changes with constraints imposed by proteins structures and physiological roles. Here, we explore strategies to more sensitively detect signatures of positive or relaxed selection by integrating evolutionary analyses with structural and functional information, using meiotic recombination proteins in four taxa--primates, rodents, birds and budding yeasts. By mapping selection rate estimates onto predicted protein structures, we characterized protein regions likely to have experienced positive selection. We further identified subtle sequence variation within protein domains that are well conserved generally because of structural constraints. To detect sequence variation masked by these constraints, we analyzed selection at structurally matched residues, comparing homologs across different lineages as well as between meiosis-specific and generalist paralogs. These approaches identified lineage- and paralog-restricted enrichment of non-synonymous substitutions that may indicate loss of functional constraints and/or adaptive innovation. Finally, we used cross-species complementation experiments in Saccharomyces cerevisiae to show that sequence variation in the pro-crossover factor MSH4 modulates recombination proficiency. We suggest that evolutionary plasticity per se is a key conserved characteristic of the meiotic recombination machinery. More generally, our approach provides a mechanistic framework to analyze protein evolution.

evolutionary biology↗

bioRxiv2024

Reconstitution of SPO11-dependent double-strand break formation

Homologous meiotic recombination starts with DNA double-strand breaks (DSBs) generated by SPO11 protein1. SPO11 is critical for meiosis in most species but the DSBs it makes are also dangerous because of their mutagenic2 and gametocidal3 potential, so cells must foster SPO11s beneficial functions while minimizing its risks4. SPO11 mechanism and regulation remain poorly understood. Here we report reconstitution of DNA cleavage in vitro with purified recombinant mouse SPO11 bound to its essential partner TOP6BL. Similar to their yeast orthologs5,6, SPO11- TOP6BL complexes are monomeric (1:1) in solution and bind tightly to DNA. Unlike in yeast, however, dimeric (2:2) assemblies of mouse SPO11-TOP6BL cleave DNA to form covalent 5' attachments requiring SPO11 active site residues, divalent metal ions, and SPO11 dimerization. Surprisingly, SPO11 can also manifest topoisomerase activity by relaxing supercoils and resealing DNA that it has nicked. Structure modeling with AlphaFold37 illuminates the protein-DNA interface and suggests that DNA is bent prior to cleavage. Deep sequencing of in vitro cleavage products reveals a rotationally symmetric base composition bias that partially explains DSB site preferences in vivo. Cleavage is inefficient on complex DNA substrates, partly because SPO11 is readily trapped in DSB-incompetent (presumably monomeric) binding states that exchange slowly. However, cleavage is improved by using substrates that favor DSB-competent dimer assembly, or by fusing SPO11 to an artificial dimerization module. Our results inform a model in which intrinsically feeble dimerization restrains SPO11 activity in vivo, making it exquisitely dependent on accessory proteins that focus and control DSB formation so that it happens only at the right time and the right places.

biochemistry↗

bioRxiv2023

Cryo-EM structure of the Spo11 core complex bound to DNA

The DNA double-strand breaks that initiate meiotic recombination are formed by topoisomerase relative Spo11, supported by conserved auxiliary factors. Because high-resolution structural data are lacking, many questions remain about the architecture of Spo11 and its partners and how they engage with DNA. We report cryo-EM structures at up to 3.3 [A] resolution of DNA-bound core complexes of Saccharomyces cerevisiae Spo11 with Rec102, Rec104, and Ski8. In these structures, monomeric core complexes make extensive contacts with the DNA backbone and with the recessed 3-OH and first 5 overhanging nucleotide, definitively establishing the molecular determinants of DNA end-binding specificity and providing insight into DNA cleavage preferences in vivo. The structures of individual subunits and their interfaces, supported by functional data in yeast, provide insight into the role of metal ions in DNA binding and uncover unexpected structural variation in homologs of the Top6BL component of the core complex.

biochemistry↗

bioRxiv2019

Cdc14 phosphatase ensures resolution of aberrant meiotic recombination outcomes partly through activating Yen1/GEN1

Meiotic defects derived from incorrect DNA repair during gametogenesis can lead to mutations, aneuploidies and infertility. Coordinated resolution of meiotic recombination intermediates is required for crossover formation, ultimately necessary for accurate completion of both rounds of chromosome segregation. Numerous master kinases orchestrate the correct assembly and activity of the repair machinery. Although much less is known, reversal of phosphorylation events in meiosis must also be key to coordinate the timing and functionality of repair enzymes. Cdc14 is an evolutionarily conserved phosphatase required for the dephosphorylation of multiple CDK1 targets. Mutations that inactivate this phosphatase lead to meiotic failure, but until now it was unknown if Cdc14 plays a direct role in meiotic recombination. Here, we show that elimination of Cdc14 leads to severe defects in the processing and resolution of recombination intermediates, causing a drastic depletion of crossovers when other repair pathways are compromised. We also show that Cdc14 is required for correct activity and localization of the Holliday Junction resolvase Yen1/GEN1. We reveal that Cdc14 regulates Yen1 activity from meiosis I onwards, and this function is essential for crossover resolution in the absence of other repair pathways. We also demonstrate that Cdc14 and Yen1 are required to safeguard sister chromatid segregation during the second meiotic division, a late action that is independent of the earlier role in crossover formation. Thus, this work uncovers previously undescribed functions of Cdc14 in the regulation of meiotic recombination.

genetics↗