Abstract
In the pathogenic Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis, the genetic and biochemical mechanisms for initiation of DNA replication are largely unknown. In the present study, we have characterized the physical interactions between M. tuberculosis DnaA and DnaB using both in vivo methods, such as bacterial two-hybrid assays, and in vitro techniques, such as surface plasmon resonance (SPR) and Pull-down/Western blotting. The full-length N-terminus (1–206 residues) of DnaB was found to interact with DnaA, while the shorter N-terminal domain of DnaB (1–125 residues), which lacked the linker region, did not. Further SPR and electrophoretic mobility shift assays indicated that the N-terminus (1–206 residues) of DnaB also had a critical role in regulating DnaA complex formation at the origin of replication (OriC). This regulatory effect was not obviously observed for DNA substrates containing only two DnaA-boxes. This is the first report showing a physical interaction between DnaA and replicative helicase DnaB from M. tuberculosis and the role in subsequent DnaA-OriC interactions. The findings reported here further the understanding of the regulatory mechanisms for initiation of DNA replication in this important human pathogen.
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Abbreviations
- 3-AT:
-
3-amino-1,2,4-triazole
- GST:
-
glutathione-S-transferase
- NTA-chip:
-
nitrilotriacetic acid chip
- OriC:
-
origin of replication
- PCR:
-
polymerase chain reaction
- SA-chip:
-
streptavidin chip
- SPR:
-
surface plasmon resonance
- str:
-
streptomycin
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Published in Russian in Biokhimiya, 2009, Vol. 74, No. 12, pp. 1622–1630.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM09-089, July 12, 2009.
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Xie, Y., He, ZG. Characterization of physical interaction between replication initiator protein DnaA and replicative helicase from Mycobacterium tuberculosis H37Rv. Biochemistry Moscow 74, 1320–1327 (2009). https://doi.org/10.1134/S0006297909120049
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DOI: https://doi.org/10.1134/S0006297909120049