In-Vitro Helix Opening of M. tuberculosis oriC by DnaA Occurs at Precise Location and Is Inhibited by IciA Like Protein

Background Mycobacterium tuberculosis (M.tb), the pathogen that causes tuberculosis, is capable of staying asymptomatically in a latent form, persisting for years in very low replicating state, before getting reactivated to cause active infection. It is therefore important to study M.tb chromosome replication, specifically its initiation and regulation. While the region between dnaA and dnaN gene is capable of autonomous replication, little is known about the interaction between DnaA initiator protein, oriC origin of replication sequences and their negative effectors of replication. Methodology/Principal Findings By KMnO4 mapping assays the sequences involved in open complex formation within oriC, mediated by M.tb DnaA protein, were mapped to position −500 to −518 with respect to the dnaN gene. Contrary to E. coli, the M.tb DnaA in the presence of non-hydrolysable analogue of ATP (ATPγS) was unable to participate in helix opening thereby pointing to the importance of ATP hydrolysis. Interestingly, ATPase activity in the presence of supercoiled template was higher than that observed for DnaA box alone. M.tb rRv1985c, a homologue of E.coli IciA (Inhibitor of chromosomal initiation) protein, could inhibit DnaA-mediated in-vitro helix opening by specifically binding to A+T rich region of oriC, provided the open complex formation had not initiated. rIciA could also inhibit in-vitro replication of plasmid carrying the M.tb origin of replication. Conclusions/Significance These results have a bearing on the functional role of the important regulator of M.tb chromosomal replication belonging to the LysR family of bacterial regulatory proteins in the context of latency.


Introduction
Replication in eubacteria is initiated when DnaA, an initiator protein, binds to DnaA boxes located within the origin of replication (oriC) sequence [1]. Initiation of replication in E. coli proceeds with the binding of DnaA protein to oriC [2] and leads to opening of 13mer region, which is followed by entry of DnaB helicase to form the prepriming complex [3]. In many bacteria either or both the 39 and 59 flanking regions of the dnaA gene exhibit oriC activity, thereby conferring the ability to replicate autonomously. In Bacillus subtilis, both the 59 and 39 flanking regions of dnaA act as oriC [4], whereas in Mycobacterium tuberculosis (M.tb), M. bovis [5] and M. smegmatis [6,7,8], only the 39 flanking region provides oriC function. There are five DnaA-binding sites in the oriC region of E. coli, referred to as R boxes, to which both active ATP-DnaA and inactive ADP-DnaA proteins bind with equal affinity [9,10]. There are additional initiator binding sites in the oriC, region referred to as I sites, to which only DnaA-ATP can bind [11].
DnaA protein binds with nearly equal affinity to ATP and ADP. In E. coli the function of ATP appears to be allosteric and the non-hydrolysable analogue ATPcS can replace ATP in helix unwinding [12]. For opening of the DNA duplex multiple DnaA proteins, complexed with ATP, bind to oriC and melt the DNA unwinding element (DUE). ADP bound form of DnaA is inactive for replication initiation, forming an important level of regulation at the origin.
The E. coli IciA protein (Inhibitor of Chromosome Initiation) blocks initiation at very early stage in-vitro by binding specifically to A+T rich region of oriC [12,13]. Binding of IciA blocks the opening of A+T rich region mediated by DnaA and HU (Histone like protein) or integration host factor (IHF) protein and this inhibition of strand opening by IciA does not affect binding of DnaA and IHF (or HU) protein to their respective binding sites [14]. IciA contains helix turn helix motif at the N terminal region and shows homology to LysR family of prokaryotic transcription regulators [12]. IciA has also been implicated in binding to A+T rich regions within the plasmid ori sequence and the copy number of the F plasmid is increased in iciA deletion mutant [15]. IciA also shows higher binding preference for curved DNA [16]. Further, IciA is involved in regulation of nrd gene encoding ribonucleoside diphosphate reductase [17], activating dnaA gene [18] and has recently been shown to also regulate the yggA gene encoding the arginine exporter [19].
M.tb maintains itself in two physiologically distinct growth states -an active replicative state and a non-replicative persistent state [20]. In persistent state, the bacterium is metabolically active, but shows no multiplication for extended periods, only to revive later and multiply to cause infection [21]. The genetic elements responsible for the replication process in M.tb, specifically its initiation and regulation, are not known. In M.tb, the DNA fragments bearing the dnaA-dnaN intergenic region function as oriC [5]. Upon comparison of the oriC region of E. coli, M.tb and B. subtilis ( Figure 1A) it appears that E. coli has three A+T rich 13 mers [1], B. subtilis has a 27 mer [4] which is exclusively rich in A+T residues, but M.tb has only one A+T rich 15 mer region [5,22]. It should also be noted that E. coli has only 5 DnaA boxes ( Figure 1B) whereas M.tb has 13 such boxes. In addition, both E. coli and B. subtilis have DnaA-ATP boxes ( Figure 1A), however in M.tb such boxes are not present [23]. One more unusual observation reported for M.tb is the requirement of hydrolysis of ATP for rapid oligomerization of DnaA on oriC [23]. It should also be noted that E. coli possesses only five DnaA boxes, whereas M.tb has 13 presumptive DnaA box sequences that bear little sequence similarity to any of the E. coli DnaA boxes [5,8]. DnaA protein of mycobacteria has been shown to bind to at least some of these boxes [24,25]. These studies suggest that the replication origin site in M.tb is very complex thereby making it interesting to study the mechanism of DNA replication and its regulation in M.tb. Given the clinical significance of persistence within the macrophages, it is important to identify and characterize the events involved in M.tb replication initiation and the negative effectors of replication initiation. We describe the interaction between M.tb DnaA protein and the M.tb oriC, including mapping the nucleotide sequences involved in DNA opening, and the requirement of ATP hydrolysis in this process. We additionally show the ability of M.tb IciA like protein, coded by Rv1985c, to block DnaA mediated helix opening and the eventual DNA replication by specifically interacting with A+T rich sequences present within the oriC region.

Results
DnaA protein shows higher ATPase activity in the presence of supercoiled template In order to determine the preference, if any, of M.tb DnaA for a given form of DNA template, DnaA protein activity was measured in terms of ATPase activity. Recombinant DnaA protein expressed in E. coli was refolded after its purification under denaturing conditions and assayed for ATPase activity. ATPase activity was assayed either in the absence of DNA, or in the presence of linear DNA, or supercoiled pUC_OriMtb, or non-specific supercoiled template pBSK II. As could be seen from the densitometric scanning of the gel, ATPase activity in the presence of DnaA box ( Figure 2, lanes 5-8) is expectedly higher than in the absence of DNA ( Figure 2, lanes 1-4). However, ATPase activity increases significantly in the presence of supercoiled pUC_OriMtb (lanes 9-12) and pBSK II (lanes 13-16) ( Table 1). The ATPase activity is a direct function of the concentration of rDnaA protein with maximal activity at 0.8 mM after which it stabilizes. These results while confirming that the refolded rDnaA protein is enzymatically active, also confirm that DnaA has very weak intrinsic ATPase activity which however increases in the presence of supercoiled DNA independent of whether M.tb oriC is present or not.
Open complex is formed near the A+T rich repeat oriC region of M.tb is very complex and is different from its E. coli counterpart ( Figure 1). The M.tb oriC has 13 imperfect DnaA boxes, which bear little sequence homology to E. coli DnaA boxes and also lack distinct A+T rich nucleotide repeat which is however present both in E. coli and B. subtilis at the 39 end of dnaA gene, and is thought to be the site for helix opening. Given this complexity of M.tb oriC, DNA sequences involved in open complex formation were therefore mapped by primer extension analyses by KMnO 4 probing. Permanganate is a very strong oxidant and thus reacts with the base moiety of DNA. Unlike DNase I, KMnO 4 generally does not modify naked double stranded DNA. However KMnO 4 selectively oxidizes unpaired pyrimidines, especially thymine residues, in single stranded DNA and in helically distorted duplex DNA. The most reactive site of the attack is 5, 6 double bond of the thymine ring. This attack can occur either from above or below the plane of the ring. But in native B form DNA this kind of attack is strongly hindered. The susceptible bond lies within the stacked array of bases under the DNA backbone within the major groove of DNA. Thus out of plane attack is just not possible as it is hindered by both the backbone and the adjacent bases. This accounts for the high selectivity of KMnO 4 for single stranded DNA. The initial stable product of the attack on thymine is glycol (diol form). Oxidized pyrimidines prevent primer extension by the DNA polymerase beyond the modified residues. This technique is routinely used for the study of replication complexes.
For our helix-opening assay increasing amounts of DnaA protein (0.025-0.3 mg) were incubated in presence of 5 mM ATP with supercoiled pUC_OriMtb, as described. Primer SeqOriR1 annealed between position 2292 to 2320 of template strand ( Figure 3A), primer SeqOriR2 annealed between positions 2402 to 2420 of the template strand ( Figure 3C) and primer SeqOriR3 annealed at position of 240 of pUC18 ( Figure 3B). Primer extension reaction carried out using SeqOriR1 and SeqOriR2 would therefore enable read outs from bottom (downstream) while SeqOriR3 will give readouts from top (upstream). The extension products were then fractionated on a standard (6% or 15% as shown in the legend) urea sequencing gel ( Figure 3A, B and C). Helix opening could clearly be detected in the presence of 0.075 mg ( Figure 3A, lane 4) of rDnaA protein but barely when 0.025 mg or 0.050 mg ( Figure 3A, lanes 2-3) of rDnaA was used and this was evident from the presence of extension products (lane 4) of 199 nucleotides(a) and 200 nucleotides(b) corresponding to position 2500 and 2501 from the start of the dnaN gene. To further pinpoint the extent of helix opening another primer SeqOriR2 was utilized and the extension products were fractionated on 15% urea gel. As can be seen ( Figure 3C Table 1. Bacterial strains, plasmids and oligonucleotide primers used in the current study. Nucleotides in bold represent the restriction enzyme sequence appended to the primers to enable directional cloning in pET28a/pUC18 vector.

Bacterial Strains
Relevant characteristics Source/ref.   Figure 3F. To conclude, our results reveal that a 19 bp stretch of M.tb oriC becomes sensitive to KMnO 4 ( Figure 3G) thereby demonstrating, for the first time, that in M.tb the duplex opening occurs near position 2500 to 2518 (from start of dnaN gene) which lies within the A+T rich region.

IciA inhibits helix opening
IciA, in addition to other functions, is a known inhibitor of E.  Figure 3E, lane 5), is a reflection of helix opening. Once the same reaction was carried out in the presence of purified rIciA protein these extension products could not be seen ( Figure 3D

ATPase activity is essential for open complex formation
Having mapped the nucleotides (within the oriC region of M.tb) involved in opening of the duplex DNA, we investigated the requirement of ATP hydrolysis and also whether other hydrolysable and poorly hydrolysable analogues of ATP could provide the necessary energy to drive this process. The E. coli DnaA protein has a very weak ATPase activity but the intrinsic ATPase activity of M.tb DnaA promotes rapid oligomerization of DnaA on oriC and both ATP binding and ATP hydrolysis are required for rapid oligomerization of DnaA on oriC [23]. We therefore carried out helix opening reaction with 5 mM of ATP, ADP and ATPcS (Lithium salt). After oxidation with 8 mM KMnO 4 the primer extension products were fractionated as usual using 6% urea gel. Only when 5 mM ATP ( Figure 4, lane 1), but not when ADP (lane 2) or ATPcS (lane 3) was used as energy donor could rDnaA bring about helix opening as could be seen from the appearance of the expected 200/199 nucleotides primer extension product. These results while highlighting the difference between M.tb and other bacteria, directly support the role of ATP in helix opening, which is a prerequisite for replication initiation.

IciA inhibits DNA replication
Having shown the ability of rIciA to inhibit helix opening invitro, experiments were designed to assess the ability of rIciA to actually inhibit DNA replication by using a reconstituted replication system. M. bovis BCG fraction II which supports invitro replication of DNA from M.tb oriC (manuscript under preparation) was utilized. Quantitation of the radioactivity incorporated as a function of DNA replication reveals that maximal DNA synthesis occurs in the presence of 80 mg of fraction II ( Figure 5A). Therefore this concentration of fraction II was selected to test whether rIciA could inhibit DNA replication in-vitro in the presence of increasing amounts of rIciA. DNA replication assay was therefore repeated except that rIciA protein was added to the assay mix before the addition of M. bovis BCG replication competent fraction II. As could be seen in Figure 5B, DNA replication is inhibited as a direct function of its concentration. In the presence of 0.6 mg of rIciA protein only 10% replication activity could be seen. These results directly point to the ability of rIciA to act as an inhibitor of DNA replication.

IciA binds to A+T rich region of M.tb oriC
The results presented so far clearly suggest that rIciA is able to block helix opening ( Figure 3D and E) and consequent DNA replication ( Figure 5) only when it encounters the oriC sequence before DnaA protein has initiated helix opening thereby pointing to a possible ori specific DNA binding activity of rIciA protein.
M.tb oriC is located within a small patch of A+T rich sequence which was earlier mapped as the site for helix opening ( Figure 3A, B and C). Having identified the nucleotides ( Figure 3F) involved in in-vitro helix opening, oligonucleotides corresponding to this region were used to determine DNA-protein interaction involving IciA. Electrophoretic mobility shift assays were carried out using this A+T rich oriC element, rIciA and huge excess (1 mg) of poly (dI/ dC). Results clearly show that IciA protein binds to A+T rich region ( Figure 6, lanes 2-4). That this binding is specific is clearly evident from homologous and heterologous cold competition assays. Even in 100-fold molar excess of non-specific competitor DNA, the DNA-protein complex is not abrogated (Figure 6, lane 5); whereas the DNA-protein complex completely disappears in the presence of 50 fold (lane 6) and 100 fold (lane 7) molar excess of specific homologous cold competitor DNA. These results demonstrate that IciA specifically binds to A+T rich region of the oriC and the inhibitory effect of IciA on the DNA helix opening ( Figure 3D and E) and DNA replication ( Figure 5) is a likely consequence of this oriC:IciA interaction.

Discussion
Regulation of DNA replication is a very critical process mediating a switch between active and latent phase of M.tb. In the present study we focused on two critical proteins, the initiator (DnaA) and a putative inhibitor of replication (IciA), involved in DNA replication. The initiator protein, DnaA, is central for bacterial replication from chromosomal origin, oriC. In E. coli, initiation of replication starts when DnaA specifically recognizes nine base pair consensus sequence, termed DnaA box within the oriC region. E. coli has five such DnaA boxes in the oriC region, but M.tb oriC region has 13 such DnaA boxes. Also the oriC of M.tb lacks a distinct A+T rich repeats and the binding of DnaA to all 13 DnaA boxes is not simultaneous. It has been proposed that DnaA first binds to a few high affinity DnaA boxes followed by binding to low affinity DnaA boxes to form a productive DnaA oriC initiation complex [23]. This oligomerization results in a local unwinding of the DNA double helix at 2500 and 2518 relative to start of dnaN gene.
Earlier studies used P1 nuclease for mapping helix opening of a supercoiled plasmid [1,14] or KMnO 4 probing for distorted B form of DNA [26,27]. We have used potassium permanganate (KMnO 4 ) probing assay to monitor in-vitro opening of the DNA helix. Using KMnO 4 probing assay we were able to determine the locus/site of opening of the double helix in M.tb oriC. Our helix unwinding assays  reveal that DnaA mediated helix melting occurs just adjacent to a stretch of A residues within the 19 bp core of the oriC.
E. coli oriC also carries I sites, which are specific for DnaA bound to ATP. M.tb oriC however lacks such sites [23] and the orthologues/analogues of E. coli Hda, which stimulate intrinsic ATPase activity of the DnaA are also absent [28]. IHF (integration host factor) and Fis proteins which are involved in DNA bending are absent in M.tb [28]. E. coli has two histone like genes; hua and hub, whereas M.tb and M. leprae have only one hu gene denoted as hupB. The M. leprae HU protein has been shown to be associated with adhesion to Schwann cells. These arguably point to the differences in the regulation of replication in M.tb from E. coli. Our results indeed show that only the ATP bound form of DnaA is active for helix unwinding in M.tb which contrasts that observed in E. coli where dATP and the non hydrolysable analog of ATP, ATPcS as well as CTP can substitute for ATP in open complex formation, but not UTP, GTP, dTTP and dCTP [1]. Unlike in E. coli, where ATP functions allosterically [10], in M.tb ATPase activity is also required. That ATP is critical for helix opening in M.tb is further supported by the observation that mutants defective in ATP hydrolysis were not viable [23]. Mutants which can bind ATP, but are unable to hydrolyze, are functionally similar to a situation of DnaA binding to ATPcS.
DnaA -ATP in E. coli is negatively regulated by Hda protein, by a process called RIDA (Regulatory Inactivation of DnaA). Hda and the b sliding clamp subunit (b clamp) of the DNA polymerase promotes hydrolysis of ATP bound to DnaA and thus inactivate DnaA [29]. Another mechanism of regulation of initiation involves the binding of many DnaA molecules to a chromosomal locus, datA, thereby reducing the number of DnaA molecules accessible to oriC [30,31]. Both of these mechanisms perhaps do not operate in M.tb, as both hda gene and datA locus are absent. Therefore, the intrinsic ATPase activity of DnaA of M.tb may be critical in regulating replication in their absence.
The putatively identified M.tb IciA, coded by ORF Rv1985c, inhibits helix opening as seen from KMnO 4 probing experiments. By binding specifically to A+T region, as evident from EMSA (Figure 7), rIciA inhibits interaction between DnaA protein at the A+T rich region within the oriC -a process critical for helix opening in a manner similar to that seen in E. coli [14,29]. Binding of rIciA consequently also inhibits in-vitro plasmid replication ( Figure 5). DNA replication in-vitro using M. bovis BCG fraction II represents an authentic in-vitro enzyme system for studying replication involving M.tb origin. That rIciA is able to inhibit invitro DNA replication in this reconstituted system ( Figure 5) clearly points to novel and an important role of IciA in inhibiting M.tb replication.
E. coli iciA null mutants are known to be completely viable and have the same growth rate as of wild type [12]. IciA is therefore not considered as a general replication inhibitor, but is thought to act under certain specific growth conditions. In E. coli, only limited sets of growth conditions have been evaluated and IciA and several other replication origin binding proteins may act as a replication inhibitor during nutrient starvation or during sudden changes in growth rate [15]. M.tb is known to survive for extended periods during the latency phase without any replication. During this phase bacteria sense the surrounding environmental conditions and iciA may have a role in maintaining mycobacterial latency. That IciA may have a role in M.tb latency is indirectly supported by results from E. coli where the concentration of IciA protein increases 4 fold (400 dimers per cell) as cells approach stationary phase [14] and cells which have elevated levels of IciA protein exhibit a growth lag upon transfer to fresh medium [12].
Based on our results we propose a working model for helix inhibition by IciA. The supercoiled template, having A+T rich region and 13 DnaA boxes, in the presence of DnaA protein and ATP binds to these DnaA boxes and causes rapid oligomerization of the supercoiled DNA. This interaction is favored by DNA bending proteins like HU. This is followed by the generation of open complex formation (Figure 7, upper half), so that other components of DNA replication can easily be loaded. Nearly about 19 nucleotides of the oriC region are unwound by DnaA alone, which can easily be detected by KMnO4 sensitivity of this region. The end product of this series of DNA protein interactions during M.tb chromosomal DNA replication signals the advent of the bacterial activation process. In contrast, during dormancy the IciA protein binds to the A+T rich region of the oriC (Figure 7, lower half) and this binding of IciA blocks DnaA dependent helix opening of the A+T rich region, a step critical for chromosomal initiation to occur. Consequently chromosomal DNA replication remains arrested so that M.tb can stay in a dormant state. It is therefore tempting to suggest that IciA could be one of the factor(s) involved in maintaining the latent state of growth of M.tb. Direct evidence for such a role of IciA will come from M.tb iciA knockouts in an infection model and also studies monitoring the steady expression level of M.tb IciA during latency and activation phase, in a clinical setting. While these experiments are underway, we are also investigating the quantitative expression of IciA as a molecular marker for M.tb activation.

Molecular cloning
The M. tuberculosis ORF Rv1985c and Rv0001 coding for putative IciA protein and DnaA protein respectively, were PCR amplified using genomic DNA from H37Rv and primers IciAF, IciAR, DnaAF and DnaAR, carrying specific restriction enzyme sites (Table 1), by Accutaq DNA polymerase (Sigma). The amplicons thus generated were digested with Nde1/HindIII restriction enzymes and cloned into the corresponding sites of pET28a expression vector. The resultant plasmids were labeled as pETIciA and pETDnaA. For cloning intergenic region between dnaA/dnaN genes, the corresponding region was PCR amplified using MtbOriF and MtbOriR primer pair ( Table 1). The amplicon thus generated was digested with HindIII/BamH1 restriction enzyme and cloned into the corresponding site of pUC18 vector. The resultant plasmid was labeled as pUC_Or-iMtb. The authenticity of all constructs was confirmed by restriction analysis and DNA sequencing.

Purification of recombinant His tagged IciA protein
Recombinant putative IciA, coded by M.tb ORF Rv1985c, was purified from the soluble fraction of BL21 (DE3) pLysS cells transformed with pETIciA grown overnight at 18uC and induced with 0.5 mM IPTG at an OD 600 of 0.3 for the expression of recombinant protein as described earlier [32,33]. The recombinant protein was purified in buffer containing 20 mM Tris, 300 mM NaCl and 10% glycerol. The purity of the protein was confirmed by SDS PAGE. The concentration of the protein was estimated by BCA (Bichinconic acid) and the purified protein was stored at 220uC until further use.

Purification of recombinant His tagged DnaA protein
Recombinant DnaA protein was purified as described earlier [25] with minor modifications. To prevent the recombinant protein from getting complexed with ATP present in E. coli cytoplasm, which could interfere in the helix unwinding assays, the protein was denatured in buffer A [25 mM Tris acetate (pH 7.5), 250 mM NaCl, 0.1 mM EDTA, 10 mM Magnesium acetate and 10 mM b-mercaptoethanol] containing 8 M urea [23]. This was followed by sequential dialysis in 4 M, 2 M, 1 M and 0.5 M urea in buffer A containing 10% glycerol. The final dialysis buffer A contained 20% glycerol. The refolded DnaA protein, as seen on 10% SDS PAGE, was .95% pure. The protein concentration was estimated by BCA and stored at 220uC until further use.

Preparation of fraction II
In-vitro replication competent fraction II was prepared by growing M. bovis BCG Pasteur in 600 ml of 7H9 media supplemented with OADC and casitone, in 1000 ml roller bottle at 37uC to log phase as described previously [34]. It took around 6-7 days for the cells to reach log phase from 1% primary inoculum. The cells were then harvested and resuspended in buffer B [25 mM, HEPES/KOH (pH 7.6), 0.1 mM EDTA, 2 mM DTT, and 100 mM potassium glutamate] supplemented with 1 mM PMSF. The cells were disrupted by sonication and the supernatant (fraction I) was precipitated by addition of ammonium sulphate (0.34 gm per ml of supernatant) with continuous stirring. This concentration was used for ammonium sulphate precipitation as it was known that M.tb DnaA precipitates with 34% ammonium sulphate cutoff. After an additional 30 min of stirring, the suspension was centrifuged at 4uC for 30 min at 18 000 g. The pellet was resuspended in minimal volume of buffer B (fraction II) of around 600 ml and dialyzed for 50 min at 4uC against 1000 fold excess of buffer B. Protein concentration was estimated by BCA and the replication competent fraction was flash frozen in small aliquots, so as to avoid freeze thaw, and stored at 270uC until further use. Each aliquot was used only once, after subsequent thawing the left over aliquot was discarded.

ATPase activity
Reaction samples were kept on ice in 10 ml of buffer C [50 mM HEPES/KOH (pH 7.6), 0.5 mM Magnesium acetate, 2 mM DTT and 50 mM NaCl] containing 16 nM [c 32 P]ATP and increasing amounts of DnaA protein as mentioned in figure legends. After incubating the samples for 30 min at 0uC, linear DNA carrying the DnaA box or pUC_OriMtb or pBSK II was added and the reactions were further continued at 37uC for 30 min. After this ATPase activity was determined by spotting 1.0 ml aliquot of each sample on Silica gel 60F 254 thin layer chromatography plate (TLC). TLC plate was developed with chloroform: methanol: glacial acetic acid (65:15:5, v/v/v), followed by autoradiography and analyzing the image by Typhoon Variable Mode Imager and Image Quant software.

Helix opening assay and KMnO 4 probing
The standard helix opening assay (25 ml) was carried out in a buffer containing 40 mM HEPES-KOH (pH 7.5), 8 mM Magnesium acetate, 50 mM potassium glutamate, 1 mg poly dI/dC, 30% v/v glycerol, 320 mg/ml BSA and 550 ng supercoiled template (pUC_OriMtb), with indicated amounts of DnaA and or IciA (Rv1985) protein and 5.0 mM of either ATP or ADP or ATPcS (Lithium salt). The reaction mix was incubated for 30 min on ice followed by 20 min at 37uC. KMnO 4 was then added to a final concentration of 10 mM, and the reaction was further continued for 2 min at 37uC. The reaction was stopped by the addition of stop buffer (1.75 M b mercaptoethanol and 50 mM EDTA) and samples were transferred to ice. 40 ml of phenol was then added and the samples were vortexed and centrifuged at 6000 rpm for 5 min. The supernatant was then passed through SephadexG50 spin column to purify the DNA template for use in primer extension reaction.

Primer extension
10 ml of the primer extension mix included 200 mM each dNTPs, 0.04 pM 32 P end labeled primer [SeqOriR1, SeqOriR2 or SeqOriR3 (Table 1)] 0.5 mM MgCl 2 , 2% DMSO and 0.5 Units Taq DNA polymerase (SIGMA). The mixture was subjected to primer extension (SeqOriR1) in a thermocycler for 30 cycles: 94uC for 1 min, 92uC for 30 sec., 54uC for 30 sec. and 72uC for 1 min except for 5 min in the last amplification cycle. All the conditions for primers SeqOriR2 and SeqOriR3 were identical, except that annealing was carried out at 48uC and amplification at 72uC for 40 sec. The reactions were stopped by adding 2 ml of formamide sequencing dye (95% Formamide, 10 mM NaOH, 0.05% Bromophenol blue and 0.05%Xylene Cyanol FF). The samples were heat denatured for 5 min at 95uC and subjected to 6% (or 15%) polyacrylamide gel electrophoresis containing 7 M urea. The gels were dried and analyzed by Typhoon Variable Mode Imager and Image Quant software.

Assay for DNA replication
The standard reaction (20 ml), as described earlier [34], contained 40 mM HEPES.KOH (pH 7.6), 6 mM ATP, 500 mM of each GTP, CTP and UTP, 21.6 mM Creatine phosphate (Fluka), 50 mg/ml BSA, 100 mM each of dGTP, dCTP and dTTP, 50 mM dATP; 200 cpm/molar of total deoxynucleotide [a 32 P]dATP, 11 mM Magnesium acetate, 35 mg Creatine Kinase (Sigma), 550 ng supercoiled plasmid DNA(pUC_OriMtb) and 7% PEG 10,000. All reactions were assembled on ice and started by the addition of 10-80 mg of protein (Fraction II or rIciA or both as indicated in figure legends) and incubating at 30uC for 30 min. Total nucleotide incorporation was measured by determining radioactivity retained after 10% trichloroacetic acid precipitation on nylon membrane through dot blot apparatus (BioRad). All the reactions were quantitated by Typhoon Variable Mode Imager and Image Quant software.

Electrophoretic mobility shift assays
For electrophoretic mobility shift assays, synthetic complementary oligodeoxyribonucleotides OriF1 and OriR1 (Table 1) were annealed and 59 end labeled using T 4 Polynucleotide Kinase as described earlier [35,36]. The 32 P-labelled oligonucleotides were incubated with increasing concentration of IciA protein, at 30uC in binding buffer D [10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 50 mg/ml BSA, 1 mg poly dI/dC and 20% glycerol] for 30 min and the DNA-protein complex was fractionated on 5% native PAGE [0.256 TBE (22.25 mM Tris/ borate/0.25 mM EDTA)] at 150 V, 4uC for 2-3 hrs. The gels were dried and analyzed by Typhoon Variable Mode Imager and Image Quant software.