Deveryshetty Jaigeeth, Chadda Rahul, Mattice Jenna, Karunakaran Simrithaa, Rau Michael J, Basore Katherine, Pokhrel Nilisha, Englander Noah, Fitzpatrick James A J, Bothner Brian, Antony Edwin
Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO.
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT.
bioRxiv. 2023 Jun 6:2023.02.05.527205. doi: 10.1101/2023.02.05.527205.
Homologous recombination (HR) is a pathway for the accurate repair of double-stranded DNA breaks. These breaks are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Rad52 is a mediator protein that promotes HR by facilitating formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Canonically, Rad52 has been described to function by displacing RPA to promote Rad51 binding. However, , Rad51 readily forms a filament by displacing RPA in the absence of Rad52. Yet, , Rad52 is essential for HR. Here, we resolve how Rad52 functions as a mediator using single-particle cryo-electron microscopy and biophysical approaches. We show that Rad52 functions as a homodecamer and catalyzes single-position nucleation of Rad51. The N-terminal half of Rad52 is a well-ordered ring, while the C-terminal half is disordered. An intrinsic asymmetry within Rad52 is observed, where one or a few of the C-terminal halves interact with the ordered N-terminal ring. Within the C-terminal half, we identify two conserved charged patches that harbor the Rad51 and RPA interacting motifs. Interactions between these two charged patches regulate a ssDNA binding. These features drive Rad51 binding to a single position on the Rad52 decameric ring. We propose a Rad52 catalyzed single-position nucleation model for the formation of pre-synaptic Rad51 filaments in HR.
同源重组（HR）是一种精确修复双链DNA断裂的途径。这些断裂被切除以产生单链DNA（ssDNA），其被复制蛋白A（RPA）包被。Rad52是一种介导蛋白，通过促进在RPA包被的ssDNA上形成Rad51核蛋白丝来促进同源重组。传统上，Rad52被描述为通过取代RPA来促进Rad51结合从而发挥作用。然而，在没有Rad52的情况下，Rad51很容易通过取代RPA形成丝状物。然而，Rad52对于同源重组是必不可少的。在这里，我们使用单颗粒冷冻电子显微镜和生物物理方法解析了Rad52作为介导蛋白的作用方式。我们表明Rad52作为同型十聚体发挥作用，并催化Rad51的单位置成核。Rad52的N端一半是一个有序的环，而C端一半是无序的。在Rad52中观察到一种内在的不对称性，其中一个或几个C端一半与有序的N端环相互作用。在C端一半内，我们确定了两个保守的带电区域，它们包含Rad51和RPA相互作用基序。这两个带电区域之间的相互作用调节ssDNA结合。这些特征驱动Rad51结合到Rad52十聚体环上的单个位置。我们提出了一个Rad52催化的单位置成核模型，用于在同源重组中形成突触前Rad51丝。
