分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: Lysine acetylation is an important post-translational modification and is known to regulate many eukaryotic cellular processes. Little, however, is known about acetylated proteins in prokaryotes. Here, using immunoblotting, mass spectrometry and mutagenesis studies, we investigate the acetylation dynamics of the DNA repair protein Ku and its relationship with the deacetylase protein Sir2 and the non-homologous end joining (NHEJ) pathway in Mycobacterium smegmatis. We report that acetylation of Ku increases with growth, while NHEJ activity decreases, providing support for the hypothesis that acetylation of Ku may be involved in the DNA damage response in bacteria. Ku has multiple lysine sites. Our results indicate that K29 is an important acetylation site and that deficiency of Sir2 or mutation of K29 affects the quantity of Ku and its acetylation dynamics. Our findings expand knowledge of acetylation targets in prokaryotes and indicate a new direction for further research on bacterial DNA repair mechanisms.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-05
摘要: DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an alpha beta(2)epsilon polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its a subunit, Mtb DNA pol III has two potential proofreading subunits; the alpha and epsilon subunits. During DNA replication, the presence of the beta(2) clamp strongly promotes the polymerization of the alpha beta(2)epsilon replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb.