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A Sir2-like protein participates in mycobacterial NHEJ.

Li Z, Wen J, Lin Y, Wang S, Xue P, Zhang Z, Zhou Y, Wang X, Sui L, Bi LJ, Zhang XE - PLoS ONE (2011)

Bottom Line: Results from an in vitro glutathione S-transferase (GST) pull-down assay suggest that Sir2 interacts directly with LigD.Moreover, the Δsir2 strain was about 10-fold more sensitive to ionizing radiation (IR) in the stationary phase than the wild-type.Our results suggest that Sir2 may function closely together with Ku and LigD in the nonhomologous end-joining pathway in mycobacteria.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

ABSTRACT
In eukaryotic cells, repair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway is critical for genome stability. In contrast to the complex eukaryotic repair system, bacterial NHEJ apparatus consists of only two proteins, Ku and a multifunctional DNA ligase (LigD), whose functional mechanism has not been fully clarified. We show here for the first time that Sir2 is involved in the mycobacterial NHEJ repair pathway. Here, using tandem affinity purification (TAP) screening, we have identified an NAD-dependent deacetylase in mycobacteria which is a homologue of the eukaryotic Sir2 protein and interacts directly with Ku. Results from an in vitro glutathione S-transferase (GST) pull-down assay suggest that Sir2 interacts directly with LigD. Plasmid-based end-joining assays revealed that the efficiency of DSB repair in a sir2 deletion mutant was reduced 2-fold. Moreover, the Δsir2 strain was about 10-fold more sensitive to ionizing radiation (IR) in the stationary phase than the wild-type. Our results suggest that Sir2 may function closely together with Ku and LigD in the nonhomologous end-joining pathway in mycobacteria.

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IR sensitivity of sir2- or ku-deficient strains.Cultures of wild-type, Δsir2 and Δku strains of M. smegmatis were harvested in the log phase (A600 = 0.3), stationary phase (A600 = 2.5) or late stationary phase (A600>2.5). After irradiation using a 60Co source at a dose rate of 14 Gy/min, serial dilutions (10−1–10−4) of cultures were spotted (20 µl) onto 7H10 plates. The plates were incubated at 37°C for 3 days. Results presented are representative of three replicate experiments.
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pone-0020045-g006: IR sensitivity of sir2- or ku-deficient strains.Cultures of wild-type, Δsir2 and Δku strains of M. smegmatis were harvested in the log phase (A600 = 0.3), stationary phase (A600 = 2.5) or late stationary phase (A600>2.5). After irradiation using a 60Co source at a dose rate of 14 Gy/min, serial dilutions (10−1–10−4) of cultures were spotted (20 µl) onto 7H10 plates. The plates were incubated at 37°C for 3 days. Results presented are representative of three replicate experiments.

Mentions: In order to further test the role of Sir2 protein in mycobacterial NHEJ, we explored the effect of IR on wild type, sir2- or ku-deficient strains. As expected, ku-deficient cells were extremely sensitive to IR in the stationary phase (Figure 6), consistent with Ku's role in NHEJ. Moreover, sir2-deficient cells also presented marked sensitivity to IR in the stationary phase (Figure 6), confirming that Sir2 is involved in the NHEJ pathway. Furthermore, the survival curves (Figure 7) show that irradiated Δsir2 stationary phase cells had about a 10-fold reduction in viability compared to wild-type cells, further confirming that Sir2 plays a role in bacterial NHEJ. In addition, the viability of wild-type and sir2- or ku-deficient strains was affected to a similar extent when exposed to irradiation during the logarithmic phase (Figure 7). It is known that NHEJ is required for prokaryotic DSB repair in the stationary phase [17], [25]; the above evidence that the Δsir2 strain was more sensitive to IR in the stationary phase than in the log phase when compared to the wild-type further indicates that Sir2 protein plays a role in NHEJ. Taken together, these results therefore provide strong evidence supporting the hypothesis that Sir2, like Ku, is involved in the mycobacterial NHEJ pathway during the stationary phase.


A Sir2-like protein participates in mycobacterial NHEJ.

Li Z, Wen J, Lin Y, Wang S, Xue P, Zhang Z, Zhou Y, Wang X, Sui L, Bi LJ, Zhang XE - PLoS ONE (2011)

IR sensitivity of sir2- or ku-deficient strains.Cultures of wild-type, Δsir2 and Δku strains of M. smegmatis were harvested in the log phase (A600 = 0.3), stationary phase (A600 = 2.5) or late stationary phase (A600>2.5). After irradiation using a 60Co source at a dose rate of 14 Gy/min, serial dilutions (10−1–10−4) of cultures were spotted (20 µl) onto 7H10 plates. The plates were incubated at 37°C for 3 days. Results presented are representative of three replicate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102665&req=5

pone-0020045-g006: IR sensitivity of sir2- or ku-deficient strains.Cultures of wild-type, Δsir2 and Δku strains of M. smegmatis were harvested in the log phase (A600 = 0.3), stationary phase (A600 = 2.5) or late stationary phase (A600>2.5). After irradiation using a 60Co source at a dose rate of 14 Gy/min, serial dilutions (10−1–10−4) of cultures were spotted (20 µl) onto 7H10 plates. The plates were incubated at 37°C for 3 days. Results presented are representative of three replicate experiments.
Mentions: In order to further test the role of Sir2 protein in mycobacterial NHEJ, we explored the effect of IR on wild type, sir2- or ku-deficient strains. As expected, ku-deficient cells were extremely sensitive to IR in the stationary phase (Figure 6), consistent with Ku's role in NHEJ. Moreover, sir2-deficient cells also presented marked sensitivity to IR in the stationary phase (Figure 6), confirming that Sir2 is involved in the NHEJ pathway. Furthermore, the survival curves (Figure 7) show that irradiated Δsir2 stationary phase cells had about a 10-fold reduction in viability compared to wild-type cells, further confirming that Sir2 plays a role in bacterial NHEJ. In addition, the viability of wild-type and sir2- or ku-deficient strains was affected to a similar extent when exposed to irradiation during the logarithmic phase (Figure 7). It is known that NHEJ is required for prokaryotic DSB repair in the stationary phase [17], [25]; the above evidence that the Δsir2 strain was more sensitive to IR in the stationary phase than in the log phase when compared to the wild-type further indicates that Sir2 protein plays a role in NHEJ. Taken together, these results therefore provide strong evidence supporting the hypothesis that Sir2, like Ku, is involved in the mycobacterial NHEJ pathway during the stationary phase.

Bottom Line: Results from an in vitro glutathione S-transferase (GST) pull-down assay suggest that Sir2 interacts directly with LigD.Moreover, the Δsir2 strain was about 10-fold more sensitive to ionizing radiation (IR) in the stationary phase than the wild-type.Our results suggest that Sir2 may function closely together with Ku and LigD in the nonhomologous end-joining pathway in mycobacteria.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

ABSTRACT
In eukaryotic cells, repair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway is critical for genome stability. In contrast to the complex eukaryotic repair system, bacterial NHEJ apparatus consists of only two proteins, Ku and a multifunctional DNA ligase (LigD), whose functional mechanism has not been fully clarified. We show here for the first time that Sir2 is involved in the mycobacterial NHEJ repair pathway. Here, using tandem affinity purification (TAP) screening, we have identified an NAD-dependent deacetylase in mycobacteria which is a homologue of the eukaryotic Sir2 protein and interacts directly with Ku. Results from an in vitro glutathione S-transferase (GST) pull-down assay suggest that Sir2 interacts directly with LigD. Plasmid-based end-joining assays revealed that the efficiency of DSB repair in a sir2 deletion mutant was reduced 2-fold. Moreover, the Δsir2 strain was about 10-fold more sensitive to ionizing radiation (IR) in the stationary phase than the wild-type. Our results suggest that Sir2 may function closely together with Ku and LigD in the nonhomologous end-joining pathway in mycobacteria.

Show MeSH
Related in: MedlinePlus