Additive effects of SbcCD and PolX deficiencies in the in vivo repair of DNA double-strand breaks in Deinococcus radiodurans

Abstract

Orthologs of proteins SbcD (Mre11) and SbcC (Rad50) exist in all kingdoms of life and are involved in a wide variety of DNA repair and maintenance functions, including homologous recombination and nonhomologous end joining. Here, we have inactivated the sbcC and/or sbcD genes of Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced DNA double-strand breaks (DSB). Mutants devoid of the SbcC and/or SbcD proteins displayed reduced survival and presented a delay in kinetics of DSB repair and cell division following gamma-irradiation. It has been recently reported that D. radiodurans DNA polymerase X (PolX) possesses a structure-modulated 3'-to-5' exonuclease activity reminiscent of specific nuclease activities displayed by the SbcCD complex from Escherichia coli. We constructed a double mutant devoid of SbcCD and PolX proteins. The double-mutant DeltasbcCD DeltapolX(Dr) (where Dr indicates D. radiodurans) bacteria are much more sensitive to gamma-irradiation than the single mutants, suggesting that the deinococcal SbcCD and PolX proteins may play important complementary roles in processing damaged DNA ends. We propose that they are part of a backup repair system acting to rescue cells containing DNA lesions that are excessively numerous or difficult to repair.

FIG. 1.

Mutant ΔsbcD, ΔsbcC, and ΔsbcCD strains of bacteria show an increased sensitivity to mitomycin C treatment and to γ-irradiation. Strain GY9613 (wild type, filled squares), GY12908 (ΔsbcD, filled inverted triangles), GY12909 (ΔsbcC, filled diamonds), and GY12910 (ΔsbcCD, filled triangles) bacteria grown to an A₆₅₀ of 1.5 were exposed to mitomycin C (A) or γ-irradiation (B) at doses indicated on the abscissa. Strains GY12936 (wild type/p11520, squares), GY12923 (ΔsbcCD/p11520, triangle), and GY12912 (ΔsbcCD/p13002, sbcCD⁺, circles) were also exposed to γ-irradiation (C). Cell survival was measured as described in Materials and Methods.

FIG. 2.

ΔsbcCD and ΔpolX deletions are additive and confer increased sensitivity to γ-irradiation. (A) Bacterial strains GY12936 (wild type/p11520, filled squares), GY12923 (ΔsbcCD/p11520, filled circles), GY12924 (ΔsbcCD ΔpolX_Dr/p11520, filled diamonds), and GY12926 (ΔsbcCD ΔpolX_Dr/p13002 sbcCD⁺, filled triangles) and (B) GY12937 (wild type/p11559, squares), GY12931 (ΔpolX_Dr/p11559, circles), GY12925 (ΔsbcCD ΔpolX_Dr/p11559, diamonds), GY12930 (ΔsbcCD ΔpolX_Dr/p13008, polX_Dr, triangles), and GY12929 (ΔsbcCD ΔpolX_Dr/p13007 polX_c, inverted triangles) grown to an A₆₅₀ of 1.5 were exposed to γ-irradiation at doses indicated on the abscissa, and cell survival was measured as described in Materials and Methods.

FIG. 3.

Increased sensitivity of cells devoid of SbcCD or SbcCD and PolX_Dr proteins in early exponentially growing cultures. Bacterial strains GY9613 (wild type, squares), GY12910 (ΔsbcCD, circles), GY12918 (ΔsbcCD ΔpolX_Dr, diamonds), and GY12219 (ΔpolX_Dr, triangles) grown to an A₆₅₀ of 2 (panel A) or an A₆₅₀ of 0.5 (panel B) were exposed to γ-irradiation at doses indicated on the abscissa, and cell survival was measured as described in Materials and Methods.

FIG. 4.

Bacterial ΔsbcCD and ΔsbcCD ΔpolX_Dr strains show an increased delay in intact genomic DNA restoration and in cell division after γ-irradiation. Strains GY9613 (wild type, panels A and E, squares), GY12219 (ΔpolX_Dr, panels B and E, diamonds), GY12910 (ΔsbcCD, panels C and E, triangles), and GY12918 (ΔsbcCD ΔpolX_Dr, panels D and E, circles) grown to an A₆₅₀ of 1.2 were exposed to γ-irradiation at a dose of 6,800 Gy (panel E, filled symbols) or not (panel E, open symbols), diluted in 2× TGY to an A₆₅₀ of 0.2, and then incubated in 2× TGY. At different times after irradiation, aliquots were taken to prepare DNA agarose plugs, which were digested with NotI prior to analyses by pulsed-field gel electrophoresis (panels A, B, C, and D), and to measure the number of viable cells per ml of culture (panel E).