The human topoisomerase 3 alpha (TOP3A) enzyme, in conjunction with the RMI1-2 proteins, alters the topology of various DNA structures during replication, meiosis, and homologous recombination-based DNA repair processes in the nucleus. Mutations in any of these proteins lead to severe genomic and developmental defects, underscoring their essential functions. Recent studies have also revealed an RMI1-2-independent function of TOP3A in mitochondrial DNA replication. Previously, RMI1 was shown to enhance the DNA decatenation activity of TOP3A, and direct interaction was proposed to stabilize the open state of the TOP3A DNA gate to facilitate DNA strand passage. Utilizing our recently developed magnetic tweezers assays, we directly tested the role of the RMI1-2 complex in the process of TOP3A gate opening and decatenation at the single-molecule level. In agreement with previous ensemble results, our magnetic tweezers data support a stimulatory effect of RMI1 on DNA decatenation. However, in striking contrast to previous proposals, our experiments, which directly monitored gate opening/closing kinetics, indicate that RMI1-2 facilitates closing of the TOP3A gate against tension on the DNA, potentially by influencing the structural stability of the gate region. Correspondingly, in the presence of RMI1-2, the decatenation rate of TOP3A is less inhibited by tension applied to the DNA. Furthermore, additional single-molecule and ensemble experiments indicate that RMI1-2 increases the rate and processivity of DNA relaxation by enhancing DNA binding and increases the rate of DNA ligation, suggesting an allosteric effect in the catalytic site of TOP3A. Taken together, our results indicate that while TOP3A can mediate DNA relaxation and decatenation alone, in the nucleus, RMI1-2 may aid TOP3A-mediated processing of physiologically occurring, entwined, and highly strained DNA regions by alleviating the inhibitory effect of tension.
Harami et al. (Sun,) studied this question.