Phase separation properties of RPA combine high-affinity ssDNA binding with dynamic condensate functions at telomeres

作者：Vincent Spegg, Ανδρέας Παναγόπουλος, Merula Stout, Aswini Krishnan, Giordano Reginato, Ralph Imhof, Bernd Roschitzki, Petr Ćejka, Matthias Altmeyer · 发表于：Nature Structural & Molecular Biology · 年份：2023 · DOI：10.1038/s41594-023-00932-w · 被引用次数：83 · 研究领域：Genomics and Chromatin Dynamics、RNA Interference and Gene Delivery、DNA and Nucleic Acid Chemistry

RPA has been shown to protect single-stranded DNA (ssDNA) intermediates from instability and breakage. RPA binds ssDNA with sub-nanomolar affinity, yet dynamic turnover is required for downstream ssDNA transactions. How ultrahigh-affinity binding and dynamic turnover are achieved simultaneously is not well understood. Here we reveal that RPA has a strong propensity to assemble into dynamic condensates. In solution, purified RPA phase separates into liquid droplets with fusion and surface wetting behavior. Phase separation is stimulated by sub-stoichiometric amounts of ssDNA, but not RNA or double-stranded DNA, and ssDNA gets selectively enriched in RPA condensates. We find the RPA2 subunit required for condensation and multi-site phosphorylation of the RPA2 N-terminal intrinsically disordered region to regulate RPA self-interaction. Functionally, quantitative proximity proteomics links RPA condensation to telomere clustering and integrity in cancer cells. Collectively, our results suggest that RPA-coated ssDNA is contained in dynamic RPA condensates whose properties are important for genome organization and stability.