Molecular Insights Into the Structure, Function, and Stability of the DNA Polymerase Processivity Factor From...

Emergence of drug resistance in Mycobacterium tuberculosis (Mtb) calls for newer drugs and drug targets. Essential proteins such as DNA polymerase (DNAP) processivity factor, also called sliding clamp (DnaN), are indispensable for bacterial survival, and are excellent drug targets. Here, we constructed a dnaN-conditional knockout in Mycobacterium smegmatis (MsmΔdnaN) and were able to successfully complement it with Mtb DnaN (DnaN^Mtb). To explore its structure-function-stability relationship, we generated Ala-substituted mutants of the DnaN^Mtb subunit-subunit interface, and identified R115, F116, and E319 as crucial for MsmΔdnaN survival in our complementation assay. We used biophysical, biochemical, and in silico molecular dynamics simulation methods to decipher the importance of these residues. We show that mutants exist as dimers, with lesser stability than wildtype. Except F116A, the mutants are largely folded with their CD profiles similar to wildtype. We also assembled and purified Mtb Clamp Loader Complex and used it to assess DNAP processivity function of DnaN^Mtb. Our in vitro DNA synthesis data show that PolA^Mtb does not interact with DnaN^Mtb, whereas E. coli Pol-I Klenow fragment shows enhanced DNA synthesis in presence of DnaN^Mtb, which was abolished by Griselimycin, an antibiotic that inhibits clamp-DNAP interaction. Interestingly, DnaN^Mtb mutants that did not complement loss of DnaN in MsmΔdnaN also did not support enhanced DNA synthesis by Klenow, corroborating our in vivo observation. We suggest that the Mtb clamp subunit-subunit interface is crucial for maintaining structure-function-stability, and thus can be used for the targeted development of small molecule inhibitors and peptidomimetics as potent drugs against tuberculosis.