Single-Molecule FRET Reveals Two T4 Phage MR Complex Exonuclease States Regulated by the Mre11 Dimer Interface

The Mre11/Rad50 (MR) complex serves as an initial responder to double-stranded DNA breaks and is conserved across all domains of life. Rad50 possesses ATPase activity that provides energy for the nuclease activities of Mre11. To elucidate the variation in exonuclease activity across the T4 phage MR complex population, we conducted smFRET studies utilizing a dual-labeled Cy3 and Cy5 double-stranded DNA substrate. Our findings revealed that the wild-type (WT) MR complex population was predominantly divided into two distinct rates of exonuclease activity. The majority of the population (94%) exhibited rapid exonuclease activity, with an average rate exceeding 20 nucleotides per second, while the remaining fraction showed substantially slower exonuclease activity, averaging 0.50 nucleotides per second. Furthermore, we compared the activity of the WT population to a mutant complex harboring the Mre11 mutation L101D, which has previously been shown to disrupt the Mre11 dimer interface. The MR complex with the L101D mutation exhibited a much higher percentage of the population displaying slow exonuclease activity (78%), while the remaining population exhibited fast exonuclease activity at a rate similar to that of the WT population. Additional activity parameters were evaluated, such as pause times and transition amplitudes, under various enzyme concentrations, revealing further differences between the WT and L101D MR complexes. Our results provide new insights into the molecular mechanisms of MR complex exonuclease activity and suggest that the complex may adopt different conformations with distinct kinetic properties.