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Mei5-Sae3 stabilizes Dmc1 nucleating clusters for efficient Dmc1 assembly on RPA-coated single-stranded DNA
Chin-Dian Wei1, Hao-Yen Chang1,2, Chia-Hua Lu1
1Department of Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan.
Abstract:
Interhomolog recombination in meiosis requires a meiosis-specific recombinase, Dmc1. In Saccharomyces cerevisiae, the Mei5-Sae3 complex facilitates the loading of Dmc1 onto the replication protein A (RPA)-coated single-stranded DNA (ssDNA) to form nucleoprotein filaments. In vivo, Dmc1 and Mei5-Sae3 are interdependent in their colocalization on the chromosomes. However, the mechanistic role of Mei5-Sae3 in mediating Dmc1 activity remains unclear. We used single-molecule fluorescence resonance energy transfer and colocalization single-molecule spectroscopy experiments to elucidate how Mei5-Sae3 stimulates Dmc1 assembly on ssDNA and RPA-coated ssDNA. We showed that Mei5-Sae3 stabilized Dmc1 nucleating clusters with two to three molecules on naked DNA by preferentially reducing Dmc1 dissociation rates. Mei5-Sae3 also stimulated Dmc1 assembly on RPA-coated DNA. Using green fluorescent protein-labeled RPA, we showed the coexistence of an intermediate with Dmc1 and RPA on ssDNA before RPA dissociation. Moreover, the displacement efficiency of RPA depended on Dmc1 concentration, and its dependence was positively correlated with the stability of Dmc1 clusters on short ssDNA. These findings suggest a molecular model that Mei5-Sae3 mediates Dmc1 binding on RPA-coated ssDNA by stabilizing Dmc1 nucleating clusters, thus altering RPA dynamics on DNA to promote RPA dissociation.
Insights
The Mei5-Sae3 complex stabilizes DNA recombinase Dmc1 assembly on single-stranded DNA, facilitating replication protein A (RPA) displacement during meiosis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Meiosis requires interhomolog recombination mediated by the Dmc1 recombinase.
- The Mei5-Sae3 complex is crucial for loading Dmc1 onto replication protein A (RPA)-coated single-stranded DNA (ssDNA) in Saccharomyces cerevisiae.
- The precise mechanism by which Mei5-Sae3 facilitates Dmc1 activity remains incompletely understood.
Purpose of the Study:
- To elucidate the molecular mechanism by which the Mei5-Sae3 complex stimulates Dmc1 assembly on ssDNA.
- To investigate the role of Mei5-Sae3 in Dmc1-mediated RPA displacement from ssDNA.
- To understand the interplay between Dmc1, Mei5-Sae3, and RPA during meiotic recombination initiation.
Main Methods:
- Single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy.
- Colocalization single-molecule spectroscopy.
- Experiments utilizing green fluorescent protein-labeled RPA.
Main Results:
- Mei5-Sae3 stabilizes Dmc1 nucleating clusters on naked ssDNA by reducing Dmc1 dissociation rates.
- Mei5-Sae3 promotes Dmc1 assembly on RPA-coated ssDNA, leading to an intermediate state with both Dmc1 and RPA.
- RPA displacement efficiency is dependent on Dmc1 concentration and positively correlated with the stability of Dmc1 clusters.
Conclusions:
- Mei5-Sae3 stabilizes Dmc1 nucleating clusters, which is critical for efficient Dmc1 loading onto RPA-coated ssDNA.
- Mei5-Sae3 modulates RPA dynamics on ssDNA, promoting RPA dissociation to facilitate Dmc1 filament formation.
- This study proposes a molecular model for Mei5-Sae3's role in initiating meiotic recombination by stabilizing Dmc1 binding and promoting RPA removal.
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