The deinococcal DdrB protein is involved in an early step of DNA double strand break repair and in plasmid transformation through its single-strand annealing activity

Abstract

The Deinococcus radiodurans bacterium exhibits an extreme resistance to ionizing radiation. Here, we investigated the in vivo role of DdrB, a radiation-induced Deinococcus specific protein that was previously shown to exhibit some in vitro properties akin to those of SSB protein from E. coli but also to promote annealing of single stranded DNA. First we report that the deletion of the C-terminal motif of the DdrB protein, which is similar to the SSB C-terminal motif involved in recruitment to DNA of repair proteins, did neither affect cell radioresistance nor DNA binding properties of purified DdrB protein. We show that, in spite of their different quaternary structure, DdrB and SSB occlude the same amount of ssDNA in vitro. We also showed that DdrB is recruited early and transiently after irradiation into the nucleoid to form discrete foci. Absence of DdrB increased the lag phase of the extended synthesis-dependent strand annealing (ESDSA) process, affecting neither the rate of DNA synthesis nor the efficiency of fragment reassembly, as indicated by monitoring DNA synthesis and genome reconstitution in cells exposed to a sub-lethal ionizing radiation dose. Moreover, cells devoid of DdrB were affected in the establishment of plasmid DNA during natural transformation, a process that requires pairing of internalized plasmid single stranded DNA fragments, whereas they were proficient in transformation by a chromosomal DNA marker that integrates into the host chromosome through homologous recombination. Our data are consistent with a model in which DdrB participates in an early step of DNA double strand break repair in cells exposed to very high radiation doses. DdrB might facilitate the accurate assembly of the myriad of small fragments generated by extreme radiation exposure through a single strand annealing (SSA) process to generate suitable substrates for subsequent ESDSA-promoted genome reconstitution.

Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB

“…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 quaternary structure suggest a shared mechanism despite divergent evolutionary origins (12)(13)(14).…”

“…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)(4)(5)(6).…”

Structure and Cellular Dynamics of Deinococcus radiodurans Single-stranded DNA (ssDNA)-binding Protein (SSB)-DNA Complexes

“…Interestingly, localization dynamics that are very similar to those of DrSSB have been observed for an alternative D. radiodurans SSB (DdrB) that has important roles in DNA repair (47)(48)(49). Although DdrB binds ssDNA, it lacks the canonical OB DNA-binding fold shared among most SSBs.…”

The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery

“…Among the proteins that localize with the DNA uptake apparatus and are competence-induced, the absence of RecA and DprA shows the strongest phenotype (Table 3). Except in D. radiodurans that chromosomal transformation is reduced only 40- to 100-fold (Bouthier de la Tour et al, 2011, Satoh et al, 2012), the absence of RecA abolishes chromosomal transformation (reduced > 10 4 -fold). In general plasmid transformation is a RecA-independent event (2- to 4-fold reduction), but in some species (e.g., S. pneumoniae ) plasmid transformation is indirectly abolished in the recA context.…”