2021/02/20
賀！本所冀宏源老師跨領域研究團隊研究成果榮登國際期刊PNAS
Trichoderma reesei Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences
Led by Dr. Hung-Yuan Chi, Ting-Fang Wang, Hung-Wen Li
Abstract from PNAS February 23, 2021 118 (8) e2007192118; https://doi.org/10.1073/pnas.2007192118
Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.
Fig.
TrRad51 can process strand exchange with mismatched substrate. Fluorescence-based strand-exchange assay reveals a better mismatch tolerance of TrRad51 for mismatched substrate in strand exchange compared to ScRad51. (A) Schematic of the fluorescence-based strand-exchange assay. Successful strand-exchange events separate two fluorophores, resulting in increased Cy3 signal. (B) Schematics of homologous and mismatched substrates. (C) ScRad51 (I)- or ScDmc1 (II)-mediated strand exchange with homologous (blue) or mismatched (red) substrates was monitored by Cy3 emission signal at the indicated reaction time. Experimental repeats are presented as dotted curves and statistical analysis of all repeats is reported in E. (D) TrRad51-mediated strand exchange with all substrates was monitored by Cy3 emission signal at the indicated reaction time. (E) The mismatch tolerances of all recombinases with mismatched/homologous substrates were determined at 120 min. **P < 0.01; ****P < 0.0001. Data shown are average values ± SEM from three independent experiments. Statistics was performed by one-way ANOVA with Tukey’s post hoc test.
TrRad51 can process strand exchange with mismatched substrate. Fluorescence-based strand-exchange assay reveals a better mismatch tolerance of TrRad51 for mismatched substrate in strand exchange compared to ScRad51. (A) Schematic of the fluorescence-based strand-exchange assay. Successful strand-exchange events separate two fluorophores, resulting in increased Cy3 signal. (B) Schematics of homologous and mismatched substrates. (C) ScRad51 (I)- or ScDmc1 (II)-mediated strand exchange with homologous (blue) or mismatched (red) substrates was monitored by Cy3 emission signal at the indicated reaction time. Experimental repeats are presented as dotted curves and statistical analysis of all repeats is reported in E. (D) TrRad51-mediated strand exchange with all substrates was monitored by Cy3 emission signal at the indicated reaction time. (E) The mismatch tolerances of all recombinases with mismatched/homologous substrates were determined at 120 min. **P < 0.01; ****P < 0.0001. Data shown are average values ± SEM from three independent experiments. Statistics was performed by one-way ANOVA with Tukey’s post hoc test.