Abstract
Smc5/6 is an essential genome maintenance complex that interacts with double-stranded (ds) DNA, single-stranded (ss) DNA, and ss-dsDNA junctions. DNA association underlies Smc5/6’s functions in managing intermediates generated during genome replication and repair. However, the mechanisms of this activity are not fully understood. Here, we report a single-molecule study examining Smc5/6 association with a dsDNA substrate containing a ssDNA gap with defined 3’ and 5’ junctions. We found that Smc5/6 associates with both 3’ and 5’ junctions but prefers the 3’ junction in the presence of the ssDNA-binding complex RPA. Further, Smc5/6’s junction association frequency and dwell time are regulated by two non-SMC subcomplexes and DNA binding residues of Smc6. Moreover, Smc5/6 prefers binding to junction sites free of the sliding clamp PCNA over those occupied with it. These results suggest that Smc5/6 utilizes its multiple structural modules to associate with junction sites in coordination with other genome maintenance factors.
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Data availability
Kymographs used for analysis are available via Zenodo at https://zenodo.org/records/1779265868. Source data are provided with this paper.
Code availability
All specified scripts used to process and analyze C-trap data can be accessed on the LUMICKS Harbor site (“CTrapVis”, https://github.com/lumicks/harbor/tree/main/Visualization/C-Trap%20.h5%20File%20Visualization%20GUI)69.
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Acknowledgements
We thank John Watters in the Shixin Liu Laboratory for technical support, Sophia Park in the Xiaolan Zhao Laboratory for helpful comments. J.T.C. was supported by an NCI F30 fellowship (F30CA275379). J.T.C. and B.J.K. were supported by a Medical Scientist Training Program grant to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program (T32GM152349) from the National Institutes of Health. V.M.B. and J.Z. acknowledge support from a training grant from NIGMS awarded to the Molecular Biophysics Program at Weill Cornell Graduate School (T32GM132081). G.N.L.C. was supported by an NIH F31 fellowship (F31MH132306). M.E.O. was supported by NIH (R01GM115809) and Howard Hughes Medical Institute. X.Z. was supported by NIH (R35GM145260) and Memorial Sloan-Kettering Cancer Center Core Grant P30 CA 008748?. S. Liu was supported by the Alfred P. Sloan Foundation, the Marlene Hess Center for Research in Women’s Health and Biomedicine at Rockefeller University, and NIH (R01GM149862).
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X.Z. and S.Liu oversaw the project. J.T.C., V.M.B., and G.N.C. performed single-molecule experiments and analyzed the data. S.Li, V.M.B., and J.Z. prepared protein constructs and performed biochemical experiments. B.J.K. assisted with single-molecule experiments. M.E.O. and E.C.B. provided key reagents. J.T.C., V.M.B., X.Z., and S.Liu wrote the paper with inputs from all authors.
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Chang, J.TH., Miller-Browne, V., Chua, G.N.L. et al. Molecular determinants of Smc5/6 association with DNA junctions. Nat Commun (2026). https://doi.org/10.1038/s41467-025-67999-5
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DOI: https://doi.org/10.1038/s41467-025-67999-5
