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-Structure paper
Title | Structure of RADX and mechanism for regulation of RAD51 nucleofilaments. |
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Journal, issue, pages | bioRxiv, Year 2023 |
Publish date | Sep 20, 2023 |
Authors | Swati Balakrishnan / Madison Adolph / Miaw-Sheue Tsai / Kaitlyn Gallagher / David Cortez / Walter J Chazin / |
PubMed Abstract | Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of ...Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed ssDNA. To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here we present a structure-based investigation of RADX's mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined by cryo-electron microscopy (EM) from maps in the 2-3 Å range. The structure reveals the molecular basis for RADX oligomerization and binding of ssDNA binding. The binding of RADX to RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the growing end of RAD51 filaments. |
External links | bioRxiv / PubMed:37786681 / PubMed Central |
Methods | EM (single particle) |
Resolution | 2.92 - 3.16 Å |
Structure data | EMDB-41939, PDB-8u5y: EMDB-42156: RADX trimer locally refined map 1 EMDB-42157: RADX trimer locally refined map 2 EMDB-42162: RADX trimer consensus map |
Source |
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Keywords | DNA BINDING PROTEIN/DNA / oligomer OB-fold / RAD51 regulator / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex |