The Partners Of MutM Detected In Mycobacterium Smegmatis By Tandem Affinity Purification

MutM protein and its functional homolog protein in eukaryote OGG1 are DNA glycosylase, and they play a very important role in base excision repair (BER). MutM and OGG1 are bifunctional glycosylases, not only can identify DNA lesions, but also can excise the damage base. Many kinds of DNA damage can be identified and catalyzed by them, especially 8-oxoguanine (8-oxoG) which is generated by reactive oxygen species (ROS) and is one of most common and mutagenic oxidative DNA damage. MutM and OGG1 play a key function in 8-oxoG repair and the functional mechanism of them has been well known. However, the detailed mechanisms of some other damage repair pathway which MutM and OGG1 take part in are still under ignorance. Recent study has shown that E. coli MutM protein and its functional homolog protein in S.cerevisiae OGG1 can specifically bind C/C mismatches in DNA that cannot be efficiently corrected by mutHLS mismatch and nucleotide excision repair pathways individually. However, neither MutM nor OGG1 exert any detectable incision activity on C/C substrates in vitro. In addition, the mechanism of Escherichia coli MutM suppressing illegitimate recombination is yet under unclear. In a word, the pathways MutM involving in are necessary to study in depth.Our work mainly focused on the research into mechanism of the pathways MutM involving in and exploring the novel pathways especially for C/C mismatches repair in M. smegmatis. To explore new factors of MutM, we chose the protein of MutM as bait, knocked-in a TAP (protA/CBP) tag into the C terminal of MutM in the M. smegmatis genome by the recently developed mycobacterial recombineering system, and used the method of tandem affinity purification (TAP) in proteomics screen for M. smegmatis proteins that interact with MutM. After SDS-PAGE separation and silver staining, the following mass spectrometry identification revealed a novel MutM-binding protein of UvrA. This interaction was reconfirmed by SBP pull-down and far-western blot in vitro. There has been evidence that MMR and NER cooperate in the repair of several types of lesion such as C/C mismatches and UV damage. In addition, mutHLS mismatch repair system has not been found so far in Mycobacterium neither M. smegmatis nor M. tuberculosis, as well as now we find and prove the interaction between MutM and UvrA, therefore we speculate that BER and NER cooperate in some lesions repair in mycobacterial like MMR and NER in eukaryote.Recent study has shown that NER of eukaryote takes part in C/C mismatches repair. C/C mismatches authentically are helix-distorting lesions generally removed by NER. In addation, MutM can bind C/C mismatch in E. coli. Thus we speculate that MutM and some proteins cooperate in C/C mismatches repair. This study found and successfully validated the interaction between MutM and UvrA, which provided a new view to study cooperating of BER and NER.