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In-vitro helix opening of M. tuberculosis oriC by DnaA occurs at precise location and is inhibited by IciA like protein.

Kumar S, Farhana A, Hasnain SE - PLoS ONE (2009)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Biology, CDFD, Hyderabad, India.

ABSTRACT

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 KMnO(4) 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 (ATPgammaS) 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.

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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.
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pone-0004139-g001: 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.

Mentions: 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.


In-vitro helix opening of M. tuberculosis oriC by DnaA occurs at precise location and is inhibited by IciA like protein.

Kumar S, Farhana A, Hasnain SE - PLoS ONE (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2607554&req=5

pone-0004139-g001: 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.
Mentions: 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.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Biology, CDFD, Hyderabad, India.

ABSTRACT

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 KMnO(4) 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 (ATPgammaS) 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.

Show MeSH
Related in: MedlinePlus