Mutational analysis of Mei5, a subunit of Mei5-Sae3 complex, in Dmc1-mediated recombination during yeast meiosis.

Interhomolog recombination in meiosis is mediated by the Dmc1 recombinase. The Mei5-Sae3 complex of Saccharomyces cerevisiae promotes Dmc1 assembly and functions with Dmc1 for homology-mediated repair of meiotic DNA double-strand breaks. How Mei5-Sae3 facilitates Dmc1 assembly remains poorly understood. In this study, we created and characterized several mei5 mutants featuring the amino acid substitutions of basic residues. We found that Arg97 of Mei5, conserved in its ortholog, SFR1 (complex with SWI5), RAD51 mediator, in humans and other organisms, is critical for complex formation with Sae3 for Dmc1 assembly. Moreover, the substitution of either Arg117 or Lys133 with Ala in Mei5 resulted in the production of a C-terminal truncated Mei5 protein during yeast meiosis. Notably, the shorter Mei5-R117A protein was observed in meiotic cells but not in mitotic cells when expressed, suggesting a unique regulation of Dmc1-mediated recombination by posttranslational processing of Mei5-Sae3.

The role of conserved amino acid residues of Sae3 in Mei5-Sae3 complex for Dmc1 assembly in meiotic recombination.

Meiotic recombination between homologous chromosomes is promoted by the collaborative action of two RecA homologs, Rad51 and meiosis-specific Dmc1. The filament assembly of Dmc1 is promoted by meiosis-specific Mei5-Sae3 in budding yeast. Mei5-Sae3 shows sequence similarity to fission yeast Sfr1-Swi5, which stimulates DNA strand exchanges by Rad51 as well as Dmc1. Sae3 and Swi5 share a conserved motif with the amino acid sequence YNEI/LK/RD. In this study, we analyzed the role of the YNEL residues in the Sae3 sequence in meiotic recombination and found that these residues are critical for Sae3 function in Dmc1 assembly. L59 substitution in the Sae3 protein disrupts complex formation with Mei5, while Y56 and N57 substitutions do not. These observations reveal the differential contribution of conserved YNEL residues to Sae3 activities in meiotic recombination.

Design and Synthesis of Styrenylcyclopropylamine LSD1 Inhibitors.

Leveraging the catalytic machinery of LSD1 (KDM1A), a series of covalent styrenylcyclopropane LSD1 inhibitors were identified. These inhibitors represent a new class of mechanism-based inhibitors that target and covalently label the FAD cofactor of LSD1. The series was rapidly progressed to potent biochemical and cellular LSD1 inhibitors with good physical properties. This effort resulted in the identification of 34, a highly potent (<4 nM biochemical, 2 nM cell, and 1 nM GI50), and selective LSD1 inhibitor. In-depth kinetic profiling of 34 confirmed its covalent mechanism of action, validated the styrenylcyclopropane as an FAD-directed warhead, and demonstrated that the potency of this inhibitor is driven by improved non-covalent binding (K I). 34 demonstrated robust cell-killing activity in a panel of AML cell lines and robust antitumor activity in a Kasumi-1 xenograft model of AML when dosed orally at 1.5 mg/kg once daily.