Deleting the 2B domain of mycobacterial UvrD1 unmasks a latent helicase activity in vitro but effaces UvrD1 activity in nucleotide excision repair in vivo.

Mycobacterial UvrD1 is a DNA-dependent ATPase motor that participates as a monomer in nucleotide excision repair (NER). UvrD1 per se is a feeble helicase and its helicase activity is not required for NER. UvrD1, like other superfamily-1 ATPases, consists of 1A and 2A domains that engage ATP•Mg2+. The 2A domain is punctuated by an inserted 2B domain that has been proposed to either couple ATP hydrolysis to duplex unwinding or function as an autoinhibitor of unwinding that is overcome by homodimerization. Here, we designed and characterized UvrD1-∆2B, in which the entire 2B domain is internally deleted, that enabled an evaluation of the role of the 2B domain in UvrD1 function. Removal of the 2B domain elicited a gain of helicase activity, thereby implicating the 2B domain as an impediment to UvrD1's latent duplex unwinding capacity. We report that (i) UvrD1-∆2B is unable to fulfill UvrD1's role in NER; (ii) a merodiploid uvrD1+  uvrD1-∆2B strain is as defective in NER as a ∆uvrD1 strain; and (iii) inducible overexpression of uvrD1-∆2B in wild-type cells slows growth. The latter two effects of UvrD1-∆2B are eliminated by an ATPase/helicase-inactivating mutation. We surmise that unbridled UvrD1 helicase activity is detrimental to mycobacterial physiology.