Substrate selectivity and RPA-mediated regulation of Topoisomerase3-Rmi1 decatenation

Mutations in the RecQ helicase BLM, which cause the cancer-prone disorder Bloom syndrome, lead to a high frequency of sister chromatid exchanges (SCEs). This phenotype is attributed to defects in processing double Holliday Junctions (dHJs) formed during homologous recombination. The BLM-Topo IIIalpha-RMI1/2 complex (Sgs1-Top3-Rmi1 [STR] in S. cerevisiae ) is critical for resolving dHJs into non-crossover products, thereby suppressing SCEs. This dissolution requires the Sgs1 helicase to drive convergent junction migration, creating a hemicatenane that is decatenated by the Topoisomerase III (Top3)-Rmi1 subcomplex in the model organism, S. cerevisiae .However, the precise molecular mechanism of this final strand passage is poorly understood. One of the key ambiguities is the role of Replication Protein A (RPA), the universal ssDNA-binding protein. Previous studies have shown contradictory findings, with some reporting that RPA inhibits TopoIIIalpha decatenation, while others-using different methodologies-suggest it promotes the reaction. Furthermore, the specific substrate requirements of Top3-Rmi1, such as sequence or polarity preference, remain uncharacterized.To elucidate the role of RPA and define the substrate requirements for Top3-Rmi1, we devised in vitro decatenation assay using a single-strand catenane substrate. This system allowed us to make two key discoveries: First, we reveal that Top3-Rmi1 activity has selectivity for both the sequence and polarity of the DNA substrate. Second, we propose that RPA plays a sophisticated regulatory role, acting to guide the strand passage activity of Top3. This model suggests RPA ensures that Top3 cleaves DNA only when the substrate is properly configured for productive strand passage and resealing.Furthermore, we used a key tool, Top3-W77A, a mutant we identified that exhibits an ~10-fold lower DNA binding affinity. We found that this mutant's decatenation activity is significantly diminished in the presence of RPA, suggesting its weakened DNA affinity makes its substrate more difficult to access when the DNA is coated by RPA. We used this mutant as an in vivo probe to determine the physiological involvement of RPA in two key STR-dependent pathways: double Holliday junction dissolution and replication fork template switching. Specifically, we are probing its function in defined genetic backgrounds: a rad18 deletion to assess its role in replication template switching, and an sgs1 deletion to determine the relative contributions of Sgs1.