Researchers reveal key mechanism in regulating DNA recombination

Meiotic recombination generates genetic diversity and promotes proper chromosomal segregation of parental chromosomes. This process requires a series of recombinases polymerized on single-stranded (ss) DNAs, called the nucleoprotein filament, to undergo homology search and strand exchange between homologous DNAs.

In Saccharomyces cerevisiae meiosis, programmed DNA double-strand breaks (DSBs) are formed by Spo11 to generate 3′ ssDNA tails. Once formed, ssDNA overhangs are rapidly bound by the abundant high-affinity ssDNA-binding protein, Replication Protein A (RPA), to protect these ssDNAs from nucleolytic degradation or the formation of higher-order DNA structures.

RPA-coated ssDNA substrates are distinguished from bare ssDNA substrates due to the high affinity of RPA for ssDNA; therefore, the recombination mediator Mei5-Sae3 protein complex is required for the binding of recombinases on RPA-coated ssDNA. However, the mechanistic role of Mei5-Sae3 in mediating Dmc1 activity remains unclear.

To investigate how Mei5-Sae3 stimulates Dmc1 to displace RPA and form nucleoprotein filaments, the research team from NTU Chemistry, NTU IBS and Osaka University used biochemical protein purification techniques and single-molecule FRET and Colocalization Single-Molecule Spectroscopy (CoSMoS). techniques to capture real-time binding of Dmc1 and dissociation of RPA on individual DNA with exceptional temporal resolution.

Their results are published in the journal Nucleic acid research.

Unlike traditional biological approaches, which mainly look at the final equilibrium products of the ensemble reactions, single-molecule methods could elucidate the contributions of individual biochemical steps of individual molecules, revealing temporary intermediate states that could provide insight into how the reaction proceeds .

The result showed that Mei5-Sae3 stabilized Dmc1 nucleating clusters with 2–3 molecules on naked DNA by preferentially reducing Dmc1 dissociation rates. Mei5-Sae3 also stimulated the assembly of Dmc1 on RPA-coated DNA.

Using GFP-tagged RPA, the coexistence of an intermediate containing Dmc1 and RPA on ssDNA was observed before RPA dissociation. Furthermore, the displacement efficiency of RPA was dependent 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, thereby influencing RPA dynamics on DNA to promote RPA dissociation.

The study contributes the first-ever reported detailed molecular model for this unique mediator protein Mei5-Sae3, elucidating how a mediator-recombinase interaction can drive recombinase assembly on RPA-coated ssDNA.