About one-third of the human population is estimated to be infected with Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis.1 During infection, the host immune response exposes Mtb to reactive oxygen species and reactive nitrogen intermediates that can lead to DNA damage.2, 3 This damage occurs in the form of base modifications, single-strand breaks, and double-strand breaks (DSB).4, 5 While single-strand breaks can be bypassed by replication and transcription machinery, specialized enzymes are required to repair DSBs.6, 7 Mycobacteria has three known DSB repair pathways: single-strand annealing (SSA), homologous recombination (HR), and nonhomologous end joining (NHEJ).8, 9 Repair can proceed through SSA if repeating homologous sequences are available on each side of the break. Resection leads to complementary single strands that can anneal and subsequent flap removal and end-joining lead to repair with deletion of the resected sequences.8 HR is a high-fidelity DNA repair pathway but can only function when a second intact DNA copy is present; for example, during or after replication and before division.10, 11 NHEJ can either be error-free (if the ends are sealed directly) or mutagenic depending on how the DNA ends are processed prior to ligation.9 Since NHEJ acts as the major pathway in non-replicating cells, it may be of significant relevance in persistence and pathogenesis of Mtb.12, 13, 14

Prokaryotic NHEJ is accomplished through the action of two proteins: Ku and Ligase D (LigD). Bacterial Ku proteins are ∼30 kDa, form homodimers, bind double-stranded DNA, and are responsible for identifying, binding, and protecting DSBs.15, 16 The core domain of bacterial Ku is homologous to that of the eukaryotic Ku70:Ku80 heterodimer and is highly conserved among bacterial Ku’s from different species. Each core domain forms one-half of a dimeric ring-like structure that encircles DNA.17, 18, 19 In contrast, the C-terminal domain (CTD) is unique to bacterial Ku and consists of 20–25 conserved amino acids and an extended region of basic residues of varying lengths (i.e., 14 in Mtb to 40 in M. smegmatis). The conserved sequence is important for recruiting LigD to DNA ends and the extended basic region has also been implicated in DNA binding and threading.20

DNA helicases play varied roles in DNA repair including unwinding duplex DNA, removing damaged ssDNA, stimulating the resection of blunt-ended substrates, and removing DNA-bound proteins.21, 22, 23, 24 Many bacterial helicases like UvrD, UvrD1, Rep, and PcrA, and eukaryotic helicases and helicase complexes like TFIIH, FANCJ, and WRN are involved in DNA repair pathways.25, 26, 27, 28, 29, 30 Mtb UvrD1 belongs to the UvrD/PcrA subgroup of SF1 super-family helicases and consists of two RecA-like domains (1A and 2A) with two accessory domains (1B and 2B). Members of this subgroup have been shown to unwind DNA as dimers; monomers are ssDNA translocases, but can become helicases through interactions with accessory factors.31, 32, 33, 34, 35, 36, 37, 38, 39 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

Studies performed prior to the elucidation of the redox-dependent dimerization and activation of UvrD1 showed that Mtb Ku stimulates the helicase activity of UvrD1 and provided evidence for protein–protein interactions between Ku and the CTD of UvrD1.43, 44 Here we show that Ku specifically stimulates the helicase activity of UvrD1 monomers, but only under multi-round binding conditions. This activity is slow (∼100-fold slower compared to the dimer) and inefficient (∼15% fraction of DNA substrates are unwound in the presence of saturating Ku). Furthermore, removing the C-terminal Tudor domain of UvrD1 abrogates the Ku-dependent activation. Lastly, Mtb Ku coats the dsDNA region of our unwinding substrates even in the presence of 3′ single-stranded overhangs. Ku binding is cooperative and dependent on magnesium and Ku-stimulated UvrD1 unwinding occurs even when multiple Ku dimers are bound to the DNA. These data serve to define the molecular mechanism for Ku-based activation of UvrD1 while quantitatively comparing the slow Ku-stimulated activity of monomeric UvrD1 in multi-round conditions to the rapid and efficient single-round (i.e., processive) activity of oxidatively formed dimers. These results highlight a possible role of UvrD1 monomers in remodeling and processing Ku-bound DNA during DSB repair.