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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

Identification of Sir2 as a Ku-binding protein.(A) Ku (TAP-tagged at the C-terminus) was purified by affinity chromatography with Protein A and calmodulin. Protein complexes were visualized by silver staining after separation by SDS-PAGE. Several specific bands were excised and subjected to mass spectrometry. Lane 1, the TAP tag alone as a control; lane 2, TAP-tagged Ku complex; M, Protein molecular weight marker. (B) The Sir2 peptides were identified by mass spectrometry.
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pone-0020045-g002: Identification of Sir2 as a Ku-binding protein.(A) Ku (TAP-tagged at the C-terminus) was purified by affinity chromatography with Protein A and calmodulin. Protein complexes were visualized by silver staining after separation by SDS-PAGE. Several specific bands were excised and subjected to mass spectrometry. Lane 1, the TAP tag alone as a control; lane 2, TAP-tagged Ku complex; M, Protein molecular weight marker. (B) The Sir2 peptides were identified by mass spectrometry.

Mentions: To identify Ku binding partners, we performed TAP in M. smegmatis using the constructed Ku-TAP knock-in strain. After separation by SDS-PAGE, Ku binding proteins were visualized by silver staining (Figure 2) and identified by mass spectrometry. Mass spectral data were searched using SEQUEST against NCBI M. smegmatis protein database and results were filtered and displayed using the Bioworks 3.2. Of the proteins identified with mass spectrometry, only the protein with more than one peptide identified by mass spectrometry was NAD-dependent deacetylase (MSMEG_5175). Furthermore, an independent replication of the TAP experiment (Figure S3) can only identify this deacetylase protein again. So this protein was selected for subsequent experiments. Protein sequence alignment indicated that NAD-dependent deacetylase was highly homologous with Sir2 family proteins (Figure S4) and it was named MsmSir2. Moreover, the result of a phylogenetic tree analysis revealed that MsmSir2 had more homology with SIRT5 of mammalian Sir2 family than with yeast Sir2 (Figure S4). Our results suggest that MsmSir2 is a novel interaction partner of Ku in prokaryotic cells and may be involved in NHEJ in mycobacteria.


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)

Identification of Sir2 as a Ku-binding protein.(A) Ku (TAP-tagged at the C-terminus) was purified by affinity chromatography with Protein A and calmodulin. Protein complexes were visualized by silver staining after separation by SDS-PAGE. Several specific bands were excised and subjected to mass spectrometry. Lane 1, the TAP tag alone as a control; lane 2, TAP-tagged Ku complex; M, Protein molecular weight marker. (B) The Sir2 peptides were identified by mass spectrometry.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020045-g002: Identification of Sir2 as a Ku-binding protein.(A) Ku (TAP-tagged at the C-terminus) was purified by affinity chromatography with Protein A and calmodulin. Protein complexes were visualized by silver staining after separation by SDS-PAGE. Several specific bands were excised and subjected to mass spectrometry. Lane 1, the TAP tag alone as a control; lane 2, TAP-tagged Ku complex; M, Protein molecular weight marker. (B) The Sir2 peptides were identified by mass spectrometry.
Mentions: To identify Ku binding partners, we performed TAP in M. smegmatis using the constructed Ku-TAP knock-in strain. After separation by SDS-PAGE, Ku binding proteins were visualized by silver staining (Figure 2) and identified by mass spectrometry. Mass spectral data were searched using SEQUEST against NCBI M. smegmatis protein database and results were filtered and displayed using the Bioworks 3.2. Of the proteins identified with mass spectrometry, only the protein with more than one peptide identified by mass spectrometry was NAD-dependent deacetylase (MSMEG_5175). Furthermore, an independent replication of the TAP experiment (Figure S3) can only identify this deacetylase protein again. So this protein was selected for subsequent experiments. Protein sequence alignment indicated that NAD-dependent deacetylase was highly homologous with Sir2 family proteins (Figure S4) and it was named MsmSir2. Moreover, the result of a phylogenetic tree analysis revealed that MsmSir2 had more homology with SIRT5 of mammalian Sir2 family than with yeast Sir2 (Figure S4). Our results suggest that MsmSir2 is a novel interaction partner of Ku in prokaryotic cells and may be involved in NHEJ in mycobacteria.

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