A major advance in our understanding of mutagenic mechanisms in MTB was provided by the identification in the Mizrahi laboratory of ImuC as central player in DNA damage-induced muta-genesis (Boschoff et al. 2003). In addition to demonstrating that mutagenesis depends equally on each component of the three-gene cassette (imuA'-imuB/imuC ), it established unequivocally that ImuC functions as the TLS polymerase in MTB - a critical result which highlights the need to understand the structural basis for the activity of the TLS alpha subunit (Warner et al. 2010).The MTB genome contains two genes encoding catalytic ( alpha) subunits of DNA polymerase III, imuC and dnaE. Boshoff et al. (2003) showed that these subunits are not functional alternatives. Rather, DnaE1 provides the essential replica-tive polymerase activity in MTB, while ImuC - a component of the mycobacterial DNA damage (SOS) response - is dispensable, although its deletion does result in DNA damage hypersensitivity in vitro. Significantly, loss of ImuC is also associated with attenuated virulence in vivo and a greatly reduced capacity to generate drug-resistant mutants during chronic infection. Coupled with evidence of the induction of imuC expression during prolonged infection, these observations strongly imply a role for ImuC-mediated mutagenesis in the adaptive evolution of MTB within the host (Boschoff et al. 2003).
Although SOS polymerases had been implicated in mutagenesis in other organisms prior to the discovery of ImuC, that property was thought to be exclusive to DNA polymerases of the Y family. Members of the Y family probably evolved to promote mutation avoidance and damage tolerance through a specialised ability to replicate across a variety of DNA lesions - the flipside of this ability being that the very properties enabling translesion synthesis (TLS) are implicated in mutagenesis (Yang & Woodgate 2007).For example, while the active site of Y-family polymerases is sufficiently flexible to allow bypass of lesions that might distort replicative polymerase geometry, it can result in a consequent reduction in stringency on non-substrate templates such as undamaged DNA or non-cognate lesions. The association of mutagenic activity with ImuC - a C family replicative polymerase - was therefore highly significant and demanded a reconsideration of traditional concepts of DNA synthesis fidelity. In particular, it identified ImuC as the founder member of a novel family of DnaE-type DNA polymerases that catalyse TLS synthesis in Gram positive bacteria (Tippin, Pham & Goodman 2004). Elucidating the molecular mechanics of the enzymes involved in induced mutagenesis could provide valuable insights into understanding this area of research (Warner 2010).
The aim is to define key structural characteristics of the ImuC enzyme responsible for DNA damage-induced mutagenesis in MTB that will inform our understanding of mutagenic mechanisms in MTB. The aims of the study, therefore, are to express and purify su fficient quantities of ImuC protein to enable subsequent characterisation, crystallization and structural analysis.
Comparison of the structural characteristics of ImuC with the constituitive DNA polymerase DnaE could elucidate minor variations that could account for the diff erent functions and fidelities of the otherwise similar DnaE subunits. The aim is to express and purify su fficient quantities of DnaE protein to enable subsequent characterisation, crystallization, structural analysis and comparison of DnaE with ImuC.