There were a number of cross-links between the core RNA polymerase subunits and the sigma factors

This was the case for RNA polymerase in our studies. In addition to the numerous cross-links between M. smegmatis subunits that mapped to the E. coli model, providing additional information that can be useful for modelling of this essential large protein complex. We used both C-terminal and N-terminal tagging of RpoA to determine tagging at which terminus allowed more efficient purification of the RNA polymerase complex from M. bovis BCG. We found almost the same purification efficiency regardless of the placement of the eGFP tag. However, this certainly cannot be treated as absolutely true for every protein and some proteins will require a tag to be placed on a specific terminus to allow complex formation in living cells. This may be the reason for complex formation between the glycolitic enzymes TpiA and Pgk in only one combination, not the other. These enzymes were found to be closely Tenofovir Disoproxil linked in many other organisms. In Thermotoga maritima, they were found as covalently linked fusion proteins able to form a multimeric bifunctional complex. It is highly possible that Pgk requires its C-terminus to be tag- free to be able to interact with and pull down TpiA in mycobacteria. Using the nucleotide excision repair protein UvrA in M. bovis BCG model, we proved that AP–MS-based approaches are capable of detecting dynamic changes in protein complex formation under changing circumstances. Without the DNA damaging stimuli, UvrA was found to co-purify with substantial amounts of its partner UvrB and induction of DNA damage caused specific reactions to occur within the cell, with attempts to repair the damage caused by UV irradiation, resulting in temporary dissociation of the UvrAB complex. Our data also indicates the presence of possible additional factors in the damagescanning mechanism: topoisomerase I and a DNA helicase II, annotated as UvrD2. However, additional studies will have to be conducted to understand the underlying mechanisms of such interactions. One possibility would be that the DNA integrity-scanning complex requires TopoI and DNA helicase II to respectively relax and unwind the DNA during scanning. In eukaryotes, it was shown that down modulation of topoisomerase I using antisense RNA inhibits repair of UV-induced lesions. The experiments show that TopoI is actively recruited onto genomic DNA following DNA damage by UV light,PF 429242 possibly acting during pre- or post-DNA damage processing. Similar functions of topoisomerase I may be required for effective NER repair in Mycobacteria and possibly other prokaryotes. A previous study demonstrated the interaction between UvrA, UvrB and UvrD in E. coli using immunoprecipitation. Our study provides additional evidence that these proteins form a complex in prokaryotic cells. In this single step reaction, DNA-lesions caused by alkylating substances are repaired. MGMT subsequently is ubiquitylated and degraded. Therefore, the cellular activity of MGMT is directly linked to the expression level of the protein. The high DNA repair activity of tumor cells expressing active MGMT is believed to defend the tumor from the cytotoxic effects of alkylating agents.