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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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Related in: MedlinePlus

In vitro interactions between Sir2 and LigD detected by the GST pull-down assay.(A–B) GST pull-down was used to identify the interaction between Sir2 and LigD in M. smegmatis and M. tuberculosis. (A) 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-MsmLigD. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MsmLigD. (B) Glutathione sepharose beads were incubated with 2 µg of GST-MtuSir2 (lane 3) or GST (lane 2), followed by incubation with 0.2 µg of His-MtuLigD. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MtuLigD.
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pone-0020045-g004: In vitro interactions between Sir2 and LigD detected by the GST pull-down assay.(A–B) GST pull-down was used to identify the interaction between Sir2 and LigD in M. smegmatis and M. tuberculosis. (A) 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-MsmLigD. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MsmLigD. (B) Glutathione sepharose beads were incubated with 2 µg of GST-MtuSir2 (lane 3) or GST (lane 2), followed by incubation with 0.2 µg of His-MtuLigD. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MtuLigD.

Mentions: Since the core of the prokaryotic NHEJ apparatus consists of Ku and LigD in interaction with each other, the interaction between Sir2 and LigD must be addressed when determining the function of Sir2 in the NHEJ. As shown in Figure 4, the interaction between Sir2 and LigD protein was confirmed by GST pull-down.


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)

In vitro interactions between Sir2 and LigD detected by the GST pull-down assay.(A–B) GST pull-down was used to identify the interaction between Sir2 and LigD in M. smegmatis and M. tuberculosis. (A) 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-MsmLigD. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MsmLigD. (B) Glutathione sepharose beads were incubated with 2 µg of GST-MtuSir2 (lane 3) or GST (lane 2), followed by incubation with 0.2 µg of His-MtuLigD. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MtuLigD.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020045-g004: In vitro interactions between Sir2 and LigD detected by the GST pull-down assay.(A–B) GST pull-down was used to identify the interaction between Sir2 and LigD in M. smegmatis and M. tuberculosis. (A) 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-MsmLigD. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MsmLigD. (B) Glutathione sepharose beads were incubated with 2 µg of GST-MtuSir2 (lane 3) or GST (lane 2), followed by incubation with 0.2 µg of His-MtuLigD. The bound proteins were probed with an anti-His-tag antibody. Lane 1 contains 20 ng (10% of the total input) of His-MtuLigD.
Mentions: Since the core of the prokaryotic NHEJ apparatus consists of Ku and LigD in interaction with each other, the interaction between Sir2 and LigD must be addressed when determining the function of Sir2 in the NHEJ. As shown in Figure 4, the interaction between Sir2 and LigD protein was confirmed by GST pull-down.

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