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- Materials and methods
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- Supporting Information
DNA replication is a fundamental biological process that occurs in all living organisms to ensure accurate genome inheritance. In bacteria, the DnaA boxes of the origin of replication (oriC) are specifically bound to the initiator protein DnaA, which leads to the unwinding of the adjacent AT-rich region . The single-stranded templates are then exposed to recruit DnaB helicase and other proteins to form the replisome.
DnaB is a key protein in the initiation of biological processes, such as DNA replication and elongation. DnaB functions as a helicase and catalyzes the separation of DNA duplexes into single strands in an ATP-dependent manner [2-7]. The helicases of Escherichia coli DnaB, Helicobacter pylori DnaB and pea chloroplast DnaB have been extensively characterized in vitro. Moreover, DnaB undergoes protein–protein interactions with several proteins related to DNA replication. In E. coli, DnaB can interact with DnaA initiator, DnaC loading protein, single-stranded binding protein (SSB), DnaG primase, the τ-subunit of DNA polymerase and replication termination protein [8-12]. DnaB helicase has important functions in DNA replication through these interactions from initiation to termination.
SSB is an important bioactive macromolecule in DNA metabolism, particularly in DNA replication, repair and recombination. The relation between DnaB helicase and SSB has been investigated in E. coli and H. pylori [11, 12]. Previous studies found inconsistent evidence of a physical interaction between DnaB and SSB in different bacteria. E. coli SSB is considered to have an indirect or specific interaction with DnaB helicase , but evidence for their physical interaction has been shown in H. pylori . Thus, the association between helicase and SSB appears to be biologically relevant.
Mycobacterium tuberculosis is a devastating human pathogen that infects one-third of the world's population and leads to nearly 2 million deaths annually [13, 14]. This pathogen can hide in a quiescent state in the host for long periods of time without producing overt disease symptoms under favorable conditions . Although significant effort has been exerted to understand the mechanism underlying the dormancy of these bacteria, it remains unclear. The DNA replication of M. tuberculosis is reportedly responsible for the ability of tubercle bacilli to lie dormant in the host for long periods of time .
The M. tuberculosis DnaA (MtbDnaA) protein has recently been purified and characterized [17, 18]. Similar to other DnaA proteins, MtbDnaA contains four domains, among which the C-terminal domain is responsible for DNA binding. Furthermore, MtbDnaA requires two DnaA boxes for efficient binding . In E. coli, DnaC forms a helicase complex with DnaB (DnaB6–DnaC6) and helps in its loading [8, 19]. However, similar to H. pylori, M. tuberculosis does not have an obvious DnaC homolog [20-22]. The MtbdnaB gene consists of 2625 bp nucleotides and encodes 874 amino acid residues, among which 416 amino acid residues (from Leu400 to Asn815) are intein, a segment of protein that is inserted in-frame within the precursor protein and split during post-translational maturation [23, 24]. The interaction between DnaA and DnaB in M. tuberculosis has been characterized previously . However, the function of MtbDnaB in replication and its regulation remains to be characterized.
In the present study, the putative DnaB homolog from M. tuberculosis was cloned, purified and characterized. MtbDnaB was found to have both helicase activity and ssDNA-dependent ATPase activity, regulated by MtbSSB. Furthermore, unlike the DnaB homolog from E. coli and H. pylori, MtbDnaB can only use ATP or dATP as energy source.
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- Materials and methods
- Author contributions
- Supporting Information
DnaB helicases have been isolated and characterized in several prokaryotes and eukaryotes [2-7]. However, little is known about helicases in M. tuberculosis. In the current study, an intein-deleted form of M. tuberculosis DnaB (MtbDnaB) was cloned, expressed and characterized in vitro. MtbDnaB showed strong 5′ to 3′ helicase and ATPase activities. This result suggests that MtbDnaB is a functional homolog of E. coli DnaB. Furthermore, a physical and functional interaction between MtbSSB (SSB of M. tuberculosis) and MtbDnaB was also identified.
We successfully purified the intein-deleted MtbDnaB protein and investigated its helicase activity. The basic characteristic of DnaB found across various bacterial species is its helicase activity, which enables it to unwind partial duplex DNA in the presence of ATP. Similar to DnaB from E. coli or H. pylori, MtbDnaB also exhibits this characteristic, with minor differences such as utilization of NTPs and dNTPs [2, 6]. MtbDnaB can unwind partial duplex DNA in the presence of ATP but not GTP, CTP and UTP. However, ATP, GTP and CTP can also support the helicase activity of E. coli DnaB, and GTP and UTP can be co-factors of H. pylori DnaB [2, 6]. Among the four dNTPs, only the helicase activity of MtbDnaB was supported by dATP, which is the same as H. pylori DnaB but different from E. coli DnaB, in which both dATP and dCTP are utilized.
DnaB proteins also exhibit ATPase activity, which provides the energy necessary for their helicase activity. The ATPase activity of DnaB is stimulated in the presence of ssDNA [2, 6]. The ATPase activity of MtbDnaB was also determined and further found to be stimulated by ssDNA. However, the effect of such stimulation on MtbDnaB was at a significantly lower rate than that on E. coli DnaB and H. pylori DnaB. Furthermore, a titration of increasing DNA effector with various DNA effectors that differ in secondary structures was used to evaluate the ATPase activity of MtbDnaB (Fig. S7). No significant difference was found between ssDNA and other effectors. However, the differential mechanism remains unclear.
DnaB has been observed to proceed in a 5′ to 3′, 3′ to 5′ or bipolar direction during DNA duplex unwinding [2-7, 30]. The direction of DNA unwinding depends on the polarity of DnaB movement, which is 5′ to 3′ for E. coli DnaB and H. pylori DnaB [2, 6], 3′ to 5′ for pea chloroplast DnaB  or bipolar for PcrA helicase of Bacillus anthracis . The helicase activity of MtbDnaB was found to have 5′ to 3′ polarity, similar to that of E. coli DnaB and H. pylori DnaB.
DnaB interacts with many replication-related proteins, including SSB. We found evidence for the physical interaction between MtbDnaB and MtbSSB both in vitro and in vivo. The physical interaction between DnaB and SSB is not present in all bacterial species . E. coli SSB does not have direct or specific interaction with DnaB helicase , but a physical interaction between them was reported in H. pylori . In the present study, the physical interaction between MtbDnaB and MtbSSB was first shown in vitro through bacterial two-hybrid analysis and SPR assay. Such interaction was proven to exist in vivo through co-immunoprecipitation assay. An MtbSSB mutant with deleted C-terminal 20 amino acid residues showed weaker interaction with MtbDnaB compared with wild-type MtbSSB. This result suggests that an interaction exists between MtbSSB and MtbDnaB. Cadman and McGlynn  found that only 10 amino acid residues from the C-terminus of E. coli SSB are relevant for its interaction with PriA, which is a helicase in E. coli. This phenomenon can be attributed to the poor homology between the C-terminal region of M. tuberculosis and E. coli SSBs.
The functional interaction between MtbDnaB and MtbSSB was further examined in this study. We found that the helicase activity of MtbDnaB was stimulated by MtbSSB at low concentrations and inhibited at high concentrations, which is consistent with the results observed in E. coli and H. pylori [11, 12]. SSB possibly binds the ssDNA released from the annealed substrate and prevents rehybridization of the unwound fragments at low concentrations. However, at high SSB concentrations, DnaB loading may be hindered by excess multimeric SSB in the vicinity of the fork structure. This idea is supported by our data on the helicase activity of the MtbSSB mutant variant N12S/P132L. N12S/P132L exhibited decreased binding affinity with ssDNA and a wide concentration range over which it could stimulate the helicase activity of MtbDnaB. This phenomenon can be attributed to the rehybridization of the released ssDNA caused by the weak binding affinity of N12S/P132L with ssDNA (Fig. S4). Moreover, the mutant variant F21L, which showed increased binding affinity with ssDNA, showed no effect on MtbDnaB. This phenomenon can be attributed to the strong binding of F21L in the vicinity of the fork structure, thereby preventing DnaB function.
Aside from helicase activity stimulation, SSB can also promote PriA helicase loading in E. coli [27, 28]. Therefore, we determined whether or not a similar relation exists between MtbDnaB and MtbSSB. We found that the helicase activity of MtbDnaB was weaker in the presence of SSB∆C20, which has decreased interaction with MtbDnaB compared with wild-type MtbSSB. We speculate that MtbSSB also promotes MtbDnaB helicase loading. This possibility is biologically relevant in DNA replication in M. tuberculosis. E. coli DnaC can load the DnaB helicase to oriC by forming a helicase complex (DnaB6–DnaC6) . According to our sequence analysis, the dnaC gene is absent in the M. tuberculosis genome , and the loading mechanism of MtbDnaB to oriC is unclear in this pathogen. Hence, MtbSSB–MtbDnaB interactions might be critical in restarting DNA replication, as speculated in H. pylori. Furthermore, the expression of the ssb gene was increased in BCG when this bacterium was cultured in the presence of H2O2 and methyl methanesulfonate (MMS) (Fig. S8). Thus, we presumed that MtbSSB assists the loading of MtbDnaB under normal growth conditions but prevents its binding to the vicinity of the DNA replication fork under stressful growth conditions.
In summary, we characterized the function of DnaB in M. tuberculosis and confirmed that MtbDnaB is a functional homolog of E. coli DnaB, H. pylori DnaB and pea chloroplast DnaB. We also characterized the physical and functional interactions between MtbDnaB and MtbSSB, and hypothesized that MtbSSB assists the loading of MtbDnaB on the DNA replication fork in M. tuberculosis. Our findings provide novel insights into the function and regulation of DnaB in M. tuberculosis.