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.


RESULTS
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
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.
Kumar
Sandeep
S
Laboratory of Molecular and Cellular Biology, CDFD, Hyderabad, India.
Farhana
Aisha
A
Hasnain
Seyed E
SE
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002876:Chromosomes, Bacterial / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism; D051738:Origin Recognition Complex / Q000378:metabolism; D018741:Replication Origin
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D000251:Adenosine Triphosphatases