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.
Highlights
DNA double-strand breaks (DSBs) are the most lethal form of DNA damage and pose the greatest threat to genomic DNA integrity [1,2]
To search for potential components of the mycobacterial nonhomologous end-joining (NHEJ) apparatus, we used tandem affinity purification (TAP) combined with mass spectrometry to identify components of protein complexes that interact with Ku
By using overlap extension PCR combined with a mycobacterial homologue recombination system, a TAP-tag knock-in cassette was introduced at the end of the coding region of Ku
Summary
DNA double-strand breaks (DSBs) are the most lethal form of DNA damage and pose the greatest threat to genomic DNA integrity [1,2]. They are caused by a variety of endogenous cellular processes and exogenous factors. Namely homologous recombination (HR) and nonhomologous end-joining (NHEJ), have evolved to repair DSBs and maintain genetic integrity [3,4,5,6] The latter is utilized in higher eukaryotes and is a complex pathway, involving many components such as DNAPKcs, the Ku70/80 heterodimer, Ligase IV, XRCC4, Artemis, and XLF/Cernunos [7,8]. Proteins of this pathway are critical for maintaining mammalian genomic stability
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