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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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Use of GST pull-down to confirm that Sir2 interacts with Ku.(A) GST pull-down assay. Glutathione sepharose beads were incubated with 2 µg of GST-MsmSir2 (lane 3) or GST (lane 2), followed by incubation with 0.2 µg of His-MsmKu. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MsmKu. (B) GST pull-down assay was also conducted on Sir2 and Ku from M. tuberculosis. Glutathione sepharose beads bound with GST-MtuSir2 (lane 3) or GST (lane 2) were incubated with His-MtuKu. The bound proteins separated by SDS-PAGE were analyzed by Western blotting using an anti-His-tag antibody to detect His-tagged MtuKu. Lane 1 contains 20 ng (10% of the total input) of His-MtuKu.
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pone-0020045-g003: Use of GST pull-down to confirm that Sir2 interacts with Ku.(A) GST pull-down assay. Glutathione sepharose beads were incubated with 2 µg of GST-MsmSir2 (lane 3) or GST (lane 2), followed by incubation with 0.2 µg of His-MsmKu. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MsmKu. (B) GST pull-down assay was also conducted on Sir2 and Ku from M. tuberculosis. Glutathione sepharose beads bound with GST-MtuSir2 (lane 3) or GST (lane 2) were incubated with His-MtuKu. The bound proteins separated by SDS-PAGE were analyzed by Western blotting using an anti-His-tag antibody to detect His-tagged MtuKu. Lane 1 contains 20 ng (10% of the total input) of His-MtuKu.

Mentions: To further investigate the interaction between Sir2 and Ku, an in vitro GST pull-down assay was performed. A bacterially-expressed GST-MsmSir2 fusion protein was immobilized on glutathione beads and incubated with purified His-MsmKu protein. Western blotting indicated that Sir2 interacts directly with Ku (Figure 3A). Thus, the interaction observed with the TAP method can also be reconstituted in vitro. That Sir2 also interacts directly with Ku from M. tuberculosis was confirmed by the same strategy (Figure 3B).


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)

Use of GST pull-down to confirm that Sir2 interacts with Ku.(A) GST pull-down assay. Glutathione sepharose beads were incubated with 2 µg of GST-MsmSir2 (lane 3) or GST (lane 2), followed by incubation with 0.2 µg of His-MsmKu. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MsmKu. (B) GST pull-down assay was also conducted on Sir2 and Ku from M. tuberculosis. Glutathione sepharose beads bound with GST-MtuSir2 (lane 3) or GST (lane 2) were incubated with His-MtuKu. The bound proteins separated by SDS-PAGE were analyzed by Western blotting using an anti-His-tag antibody to detect His-tagged MtuKu. Lane 1 contains 20 ng (10% of the total input) of His-MtuKu.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102665&req=5

pone-0020045-g003: Use of GST pull-down to confirm that Sir2 interacts with Ku.(A) GST pull-down assay. Glutathione sepharose beads were incubated with 2 µg of GST-MsmSir2 (lane 3) or GST (lane 2), followed by incubation with 0.2 µg of His-MsmKu. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MsmKu. (B) GST pull-down assay was also conducted on Sir2 and Ku from M. tuberculosis. Glutathione sepharose beads bound with GST-MtuSir2 (lane 3) or GST (lane 2) were incubated with His-MtuKu. The bound proteins separated by SDS-PAGE were analyzed by Western blotting using an anti-His-tag antibody to detect His-tagged MtuKu. Lane 1 contains 20 ng (10% of the total input) of His-MtuKu.
Mentions: To further investigate the interaction between Sir2 and Ku, an in vitro GST pull-down assay was performed. A bacterially-expressed GST-MsmSir2 fusion protein was immobilized on glutathione beads and incubated with purified His-MsmKu protein. Western blotting indicated that Sir2 interacts directly with Ku (Figure 3A). Thus, the interaction observed with the TAP method can also be reconstituted in vitro. That Sir2 also interacts directly with Ku from M. tuberculosis was confirmed by the same strategy (Figure 3B).

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