Figure 1.
A) Alignment of A+T rich regions from E. coli, M. tuberculosis and B. subtilis.
These regions were aligned using adjacent DnaA box (shaded arrow) to A+T rich regions. Shaded boxes represent A+T rich cluster of E. coli, M. tuberculosis and B. subtilis respectively. Underlined regions in E. coli and B. subtilis represent potential DnaA-ATP boxes. L, M and R represent left, right and middle 13-mers. B) Illustration showing the organization of oriC region of M.tb and E. coli. AT represent AT rich region (rectangle) and the arrows represent DnaA boxes. The direction of arrows represents the orientation of these boxes.
Figure 2.
A) DnaA protein (2 μM) was incubated with 16 nM [γ32P]ATP for 30 min at 0°C in buffer C.
Lanes 1–4: 0, 0.4, 0.8 and 1.2 μM respectively, of DnaA protein without any DNA; lanes 5–8: 0, 0.4, 0.8 and 1.2 μM respectively of DnaA protein with DnaA box; lanes 9–12: 0, 0.4, 0.8 and 1.2 μM respectively of DnaA protein with 550 ng of pUC_OriMtb and lanes 13–16: 0, 0.4, 0.8 and 1.2 μM respectively of DnaA protein with 550 ng of pBSK II. B) The phosphate released in ATPase assay was quantified using Typhoon Variable Mode Imager and Image Quant Software (Amersham).
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.
Figure 3.
M.tb helix opening by rDnaA occurs near position −500 to −518 within the A+T rich region and this is inhibited by rIciA.
pUC_OriMtb was used as a template for helix opening in the presence of increasing amounts of rDnaA with γ32P labeled SeqOriR1 and the primer extension products were fractionated on 6% sequencing gel. (A) SeqOriR1 primer reads pUC_OriMtb from bottom. Lanes 2, 3 and 4 show KMnO4 probing in the presence of 25 ng, 50 ng and 75 ng DnaA. These and other primer extension products of various sizes were designated as 200 nt (a), 199 nt (b) and so on and are summarized in Figure 3F. (B) The upstream primer SeqOriR3 reads pUC_OriMtb from the top and anneals at position −40 of pUC18 vector backbone. The different lanes are: lane 6, no DnaA protein; lanes 7–10: 0.075, 0.1, 0.2 and 0.3 μg of rDnaA. Extension products of 79(g), 77(h), 76(i), 66(j), 65(k) and 63(l) nucleotides could be seen. (C) Primer SeqOriR2 (downstream primer) also reads pUC_OriMtb from the 3′- end. After KMnO4 modification and PCR amplification with γ32P labeled SeqOriR2, the primer extension products were fractionated on a 15% sequencing gel. Lane 12, control DNA where no DnaA protein is added; lanes 13–16: 0.075, 0.1, 0.2 and 0.3 μg of rDnaA. Extension products of 116(c), 113(d), 99(e) and 98(f) nucleotides could be seen. Non-specific extension products (ns) were also seen in all lanes even in control DnaA free lane.10 bp ladder was used as DNA molecular size marker (lane 1, 5 and 11) and shown on the left. (D) The reaction was carried out using 0.2 μg of DnaA protein. Helix opening was monitored by primer extension using SeqOriR1 on a 6% sequencing gel. The different lanes are: lane 2, without rIciA; lane 3–4: increasing amounts (0.2 μg and 0.4 μg) of IciA protein. Arrows correspond to the extension products of 200 and 199 nucleotides. (E) Primer SeqOriR3 (lanes 5–11) and SeqOriR2 (lanes 13–19) were used to monitor helix inhibition mediated by rIciA. All the lanes from 5–9 and 13–17 have 0.2 μg of DnaA protein; Lanes 6–9 and 14–17 have increasing amounts (0.2, 0.3, 0.4 and 0.5 μg) of IciA protein; lanes 10 and 18 have 0.5 μg of IciA protein; lanes 11 and 19 have no DnaA or IciA protein; and lane 12 represents 10 bp marker. Arrows on the left correspond to extension products of 79, 77, 76, 66, 65 and 63 nucleotides with primer SeqOriR3 and 113, 99 and 98 nucleotides with primer SeqOriR2. (F) The nucleotide sequence of the entire oriC region of M.tb. Letters underlined represents various primers. Amplification products obtained by primer SeqOriR1 are marked by [ ] brackets. “]” bracket represents start of primer extension product and “[” bracket represent end of the primer extension product. It could be noted that primer extension stops at T residue which is modified by KMnO4. The small letters “a” and “b” represent 200 nt and 199 nt band. Similarly the amplification products obtained by primer SeqOriR2 (direction of primer extension product is shown by arrow) are marked by {} bracket and the modified T residues “{” mapped by this primer are indicated by c, d, e and f which represent 116, 113, 99 and 98 nucleotide bands. Likewise the amplification products obtained by primer SeqOriR3 are marked by () bracket. Here “(” bracket marks the start of extension and “)” bracket marks the end of extension product. The modified T residue is shown by g, h, i, j, k and l which represent 79, 77, 76, 66, 65 and 63 bp bands respectively. Also the start of dnaN gene is indicated by an arrow. (G) KMnO4 reactive pyrimidines within the A+T rich oriC of M.tb. About 19 bp stretch of pUC_OriMtb becomes sensitive to KMnO4 modification (the reactive pyrimidines are indicated by arrow).
Figure 4.
ATP hydrolysis is essential for helix opening by rDnaA.
Helix opening assay was carried out as described earlier with SeqOriR1 as primer and the primer extension products were resolved on 6% sequencing gel. The reaction was carried out using 0.75 μg of rDnaA and 5 mM ATP (lane 1), 5 mM ADP (lane 2) and 5 mM ATP γ S (lane 3). 100 bp DNA ladder was used as a DNA molecular size marker (lane Mr). The markers on the right represent the size of the extension products.
Figure 5.
DNA replication from pUC_OriMtb is supported by M. bovis BCG crude extracts (Fraction II) and is inhibited by rIciA.
(A) DNA replication was carried out with ammonium sulphate fractionated crude cell lysate (Fraction II), as described in Materials and Methods, in the presence of increasing concentrations (0 to 80 μg) of M. bovis BCG fraction II. Lane 1, 2, 3, 4, 5, 6 and 7 represent 0, 10, 20, 40, 60 and 80 μg of fraction II. The replication products were TCA precipitated and blotted (Biorad) on a nylon membrane and after densitometric scanning (inset) the values were plotted for different fractions. (B) DNA replication mediated by M. bovis BCG extract (80 μg) was assayed in the presence of increasing concentrations (0 to 0.6 μg) of purified rIciA.
Figure 6.
rIciA binds specifically to the A+T oligonucleotide derived from the oriC region of M. tuberculosis.
Increasing amounts of IciA protein was used in electrophoretic mobility shift assays. The different lanes are: lane 1, 0 ng; lane 2, 250 ng; lane 3, 500 ng; lane 4, 750 ng ; lane 5, 100× cold non specific competitor; lane 6, 50× and lane 7 100× of cold homologous competitors. Specific DNA protein complex is indicated by an arrow. The sequence of the oligonucleotide used is given below the gel.
Figure 7.
Schematic representation of the mechanism of helix opening by DnaA at oriC and its inhibition by IciA (modified from the model proposed by Bramhill and Kornberg [37] and Madiraju et al [23]).
The ATP bound form of replication initiator DnaA protein binds to 13 DnaA boxes (darkly shaded arrow heads pointing the orientation of DnaA boxes) in M.tb oriC located between the dnaA and dnaN gene. Binding of DnaA-ATP complex to the DnaA boxes results in rapid oligomerization leading to the formation of the initiation complex (clockwise direction). Subsequently, the initiation complex gradually opens at A+T rich region which is then acted upon by a host of replication factors which finally lead to DNA replication. When IciA is present before the formation of open complex then it follows another pathway (anticlockwise direction). Here IciA protein binds to the A+T rich region of the oriC. In the presence of the DnaA protein the initiation complex is still formed however it eventually does not lead to the formation of open complex.