Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing.single-strand DNA annealing | DNA repair | Deinococcus radiodurans | crystal structure | DdrB H ow cells overcome the need to protect ssDNA from forming deleterious secondary structures and at the same time promote accurate strand annealing represents a longstanding and intriguing question in DNA repair. Single-strand DNA-binding proteins (SSBs) safeguard DNA fragments harboring singlestrand regions from misannealing at biological temperatures by binding and occluding bases from potential interaction with other strands (1). Overcoming the thermodynamic barrier of accurate annealing under biological conditions requires specialized annealing proteins able to regulate access of unpaired bases from different strands. Although single-strand annealing (SSA) proteins have been identified in organisms ranging from bacteriophage to humans, how they promote faithful annealing of DNA ends containing large single-strand regions has remained unclear.One of the best examples of single-strand annealing comes from members of the Deinococcus family of bacteria, which are renowned for their ability to withstand and accurately repair genome fragmentation, resulting in hundreds of doublestrand breaks (DSBs) (2). Up to one-third of these breaks are repaired by a RecA-independent pathway that is dependent on single-strand annealing mediated by DdrB (DNA damage response B) (3-6).DdrB was first identified from two independent analyses of the IR-induced transcriptional response of Deinococcus radiodurans (7,8). DdrB binds ssDNA (9); however, unlike SSB, DdrB promotes accurate DNA-strand annealing (5, 10), providing evidence for a role in repair by single-strand annealing. This biological role is further supported by fluorescence microscopy data demonstrating recruitment of DdrB to the nucleoid in the early stages of repair (6) and attenuation of RecA-independent single-strand annealing repair in a ΔddrB strain (5). Although computational sequence analysis has delineated three distinct superfamilies of single-strand annealing proteins (11), similarities in function and q...
The ability of Deinococcus radiodurans to recover from extensive DNA damage is due in part to its ability to efficiently repair its genome, even following severe fragmentation by hundreds of double-strand breaks. The single-strand annealing pathway plays an important role early during the recovery process, making use of a protein, DdrB, shown to greatly stimulate ssDNA annealing. Here, we report the structure of DdrB bound to ssDNA to 2.3 Å. Pentameric DdrB was found to assemble into higher-order structures that coat ssDNA. To gain further mechanistic insight into the protein's function, a number of point mutants were generated altering both DNA binding and higher order oligomerization. This work not only identifies higher-order DdrB associations but also suggests the presence of an extended DNA binding surface running along the ‘top’ surface of a DdrB pentamer and continuing down between two individual subunits of the ring structure. Together this work sheds new insight into possible mechanisms for DdrB function in which higher-order assemblies of DdrB pentamers assist in the pairing of complementary ssDNA using an extended DNA binding surface.
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