Mycobacterium tuberculosis DNA repair helicase UvrD1 is activated by redox-dependent dimerization via a 2B domain cysteine

Abstract

Mycobacterium tuberculosis (Mtb) causes tuberculosis and, during infection, is exposed to reactive oxygen species and reactive nitrogen intermediates from the host immune response that can cause DNA damage. UvrD-like proteins are involved in DNA repair and replication and belong to the SF1 family of DNA helicases that use ATP hydrolysis to catalyze DNA unwinding. In Mtb, there are two UvrD-like enzymes, where UvrD1 is most closely related to other family members. Previous studies have suggested that UvrD1 is exclusively monomeric; however, it is well known that Escherichia coli UvrD and other UvrD family members exhibit monomer–dimer equilibria and unwind as dimers in the absence of accessory factors. Here, we reconcile these incongruent studies by showing that Mtb UvrD1 exists in monomer, dimer, and higher-order oligomeric forms, where dimerization is regulated by redox potential. We identify a 2B domain cysteine, conserved in many Actinobacteria, that underlies this effect. We also show that UvrD1 DNA-unwinding activity correlates specifically with the dimer population and is thus titrated directly via increasing positive (i.e., oxidative) redox potential. Consistent with the regulatory role of the 2B domain and the dimerization-based activation of DNA unwinding in UvrD family helicases, these results suggest that UvrD1 is activated under oxidizing conditions when it may be needed to respond to DNA damage during infection.

Mycobacterium tuberculosisKu stimulates multi-round DNA unwinding by UvrD1 monomers

“…We have previously shown that Mtb UvrD1 forms a disulfide-bonded dimer under oxidative conditions via a 2B domain cysteine in each subunit and that the dimeric form is an active helicase [42]. The monomeric form of UvrD1 – generated either via reducing conditions or by mutation of cysteine 451 in the 2B domain to alanine – can bind and translocate on single-stranded DNA but lacks helicase activity under previously tested conditions [42]. As it has been reported that Ku stimulates UvrD1 helicase activity [43], we performed DNA unwinding experiments with WT UvrD1 under conditions where dimer formation is favored in the presence and absence of Ku.…”

“…This was true under both single-round and multi-round conditions at these fast timescales (Supplementary Figure S1A) . Since the previous Ku-dependent study presented only end point measurements taken after 10 minutes, we next asked whether Ku-dependent activation occurs on longer timescales compared to the rapid unwinding exhibited by dimeric UvrD1 [42].…”

“…After overnight dialysis in (50 mM Tris-HCl pH 8.0, 60 mM NaCl, 10% glycerol) the protein was passed through DEAE-Sephacryl chromatography followed by size exclusion chromatography where it eluted as a dimer. Mtb UvrD1 proteins both wild type and C-terminal deletion mutant were expressed and purified as described previously [42].…”

“…In addition, they sometimes contain a C-terminal Tudor domain which has been implicated in protein-protein interactions with binding partners [40,41]. We previously showed that Mtb UvrD1 exists as a mixture of monomers and dimers depending on redox potential [42]. Although both monomers and dimers of UvrD1 can bind and translocate on ssDNA, only the dimer formed in oxidative conditions via a specific disulfide bond between the 2B domains possesses processive helicase activity [42].…”

Deletion of rifampicin-inactivating mono-ADP-ribosyl transferase gene of Mycobacterium smegmatis globally altered gene expression profile that favoured increase in ROS levels and thereby antibiotic resister generation

“…4 E; Supplementary Dataset S1). As an indication of DNA damage in the arr -KO cells, there is also a slight but statistically significant increase in the expression of uvrD (MSMEG_1941; Supplementary Dataset S1), which is known to get induced upon DNA damage in E. coli and M. tuberculosis ( Siegel, 1983 ; Chadda et al., 2022 , respectively).…”