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bioRxiv · 10.1101/2020.08.11.247064

PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments

Li, M.·Sengupta, B.·Benkovic, S. J.·Lee, T. H.·Hedglin, M.
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Abstract

Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the PCNA sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA damaging agents and is tightly regulated to restrict TLS to damaged DNA. RPA, the major single strand DNA (ssDNA) binding protein, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18*RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination.

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Li, M., Sengupta, B., Benkovic, S. J., Lee, T. H., Hedglin, M.. 2020-08-12. PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments. https://doi.org/10.1101/2020.08.11.247064

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