Uracil DNA glycosylases (UDGs) play an important role in removing uracil from DNA to initiate DNA base excision repair. Here, we first characterized biochemically a thermostable UDG from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba UDG), and probed its mechanism by mutational analysis.The recombinant Tba UDG cleaves specifically uracil-containing ssDNA and dsDNA at 65 o C. The enzyme displays an optimal cleavage activity at 55-75 o C. Tba UDG cleaves DNA over a wide pH spectrum ranging from 4.0 to 9.0 with an optimal pH of 5.0-8.0. In addition, the Tba UDG activity is independent on a divalent metal ion; however, both Zn 2+ and Cu 2+ completely inhibits the enzyme activity. Furthermore, the Tba UDG activity is also inhibited by high NaCl concentration. Tba UDG removes uracil from DNA by the order: U≈U/G>U/T≈U/C>U/A. The mutational studies showed that both the E118A and N159A mutants completely abolish the cleavage activity and retain the compromised binding activity, suggesting that residues E118 and N159 in Tba UDG are important for uracil recognition and removal.Our work provides a basis for determining the role of Tba UDG in the base excision repair pathway for uracil repair in Thermococcus.

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The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O 2 levels, including modulating synthesis of cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates biofilm formation. While GCS proteins have been demonstrated to regulate O 2 -dependent pathways, the mechanism by which the O 2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O 2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O 2 -dependent GCS activity.identified in numerous bacteria with a variety of output domains that are regulated by the O 2 -binding signal, including methyl accepting chemotaxis protein (MCP), kinase, and diguanylate cyclase (DGC) [12,13]. In Bacillus subtilis, the MCP-containing GCS regulates aerotaxis, allowing the organism to move to its preferred location within an O 2 gradient [14]. In contrast, the DGC-containing GCS from the plant pathogen Pectobacterium carotovorum (PccGCS) regulates O 2 -dependent motility, virulence factor production, and rotting within a plant host, highlighting the importance of O 2 sensing and GCS proteins in controlling bacterial phenotypes [11].Within the GCS protein family, the most common effect of O 2 binding to the heme of the globin domain is an increase in the activity of the output domain [13]. This increase in GCS enzymatic activity often requires oligomerization of GCS monomers to yield activity of the output domains; GCS proteins with diguanylate cyclase, histidine kinase, and adenylate cylcase output domains have been shown to function as homodimers and higher order oligomers [15][16][17][18][19][20]. Because of the multi-domain organization and oligomerization of GCS proteins, questions have arisen regarding the overall structure and path of signal transduction within the protein family. ...