PMID:21968057

Citation	Bouthier de la Tour, C, Boisnard, S, Norais, C, Toueille, M, Bentchikou, E, Vannier, F, Cox, MM, Sommer, S and Servant, P (2011) 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. DNA Repair (Amst.) 10:1223-31
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 Escherichia 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 show 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.
Links	PubMed PMC3268515 Online version:10.1016/j.dnarep.2011.09.010
Keywords	Active Transport, Cell Nucleus/radiation effects; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Cell Nucleus/metabolism; Cell Nucleus/radiation effects; DNA Breaks, Double-Stranded/radiation effects; DNA Fragmentation/radiation effects; DNA Repair/genetics; DNA Repair/radiation effects; DNA, Bacterial/biosynthesis; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; DNA, Single-Stranded/biosynthesis; DNA, Single-Stranded/genetics; DNA, Single-Stranded/metabolism; Deinococcus/genetics; Deinococcus/metabolism; Deinococcus/radiation effects; Mutation; Plasmids/genetics; Protein Structure, Tertiary; Radiation Tolerance/genetics; Time Factors; Transformation, Bacterial/radiation effects
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
DEIRA:DDRB		GO:0045002: double-strand break repair via single-strand annealing	ECO:0001141: [3H]-thymidine incorporation assay evidence used in manual assertion		P		In Figure 3 Panel A, the graphs are showing the rate of DNA synthesis in wildtype strain of the bacteria and the strain with the knockout of ddrB gene. Both strains are allowed to grow normally as well as subjected to radiation. They used a [3H]Thymidine incorporation assay to measure rate of DNA synthesis. There was an increased lag phase in DNA synthesis by an hour in the knockout compared to the wildtype strain. This lag was associated with a delay in fragment reassembly shown by PFGE, Panel B. But the rate of DNA synthesis and the fragment reassembly after this lag period was very similar to wildtype. ddrB protein also has high affinity for single stranded DNA and has similar properties to E. coli SSB protein. This is likely indicates that DdrB is involved in a very early step of DNA double strand break repair that occurs prior to strand-annealing and not in annealing the newly synthesized DNA strands itself.	complete CACAO 13758

References

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