Mycobacterium tuberculosis DnaA initiator protein: purification and DNA-binding requirements

Abstract

The Mycobacterium tuberculosis oriC (the origin of chromosomal replication) region contains 13 non-perfect DnaA boxes. The M. tuberculosis initiator protein, DnaA, was overexpressed in Escherichia coli as a soluble His-tagged fusion protein. The purified protein His6MtDnaA was investigated for its binding properties to DnaA boxes from the oriC region. Gel retardation demonstrated that the DnaA from M. tuberculosis requires two DnaA boxes for efficient binding. Electron microscopy as well as DNase I footprinting showed that the His6MtDnaA protein binds to four specific regions, which correspond to the location of 11 out of 13 previously identified DnaA boxes within the M. tuberculosis oriC. Probably, in M. tuberculosis, DnaA molecules by co-operative binding of numerous ‘non-perfect’ DnaA boxes assemble along the oriC region and subsequently form a massive nucleoprotein complex.

SMC Protein-Dependent Chromosome Condensation during Aerial Hyphal Development in Streptomyces

“…An XhoI restriction site was created directly before the stop codon of smc on cosmid 7A1 by using PCR targeting directly, and then a 2,456-bp fragment of smc was cut out and subcloned into NcoI-and XhoI-restricted pET-21a carrying smc (1,135 bp). The S. coelicolor His 6 -SMC protein was isolated as described previously (43).…”

Mechanisms for initiating cellular DNA replication

“…The mechanics and control of these proteins are complex. Some factors and their activities are conserved across all three domains of life associate with a single DnaA box; instead, binding requires that multiple repeats be present in target substrates (31). This variation, coupled with the distinct arrangement of DnaA boxes among different oriCs, suggests that DnaA orthologs are fine-tuned to act only on their cognate origin.…”

Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis

“…Knowledge regarding the steps of the mycobacterial cell cycle (replication, chromosome segregation and cell division) seems to be critical for understanding the mechanisms that are responsible for the transition from an active to a non-replicative persistent state (and vice versa) of pathogenic mycobacteria, particularly M. tuberculosis. While initiation of chromosome replication Qin et al, 1999;Rajagopalan et al, 1995;Zawilak et al, 2004) and cell division (FtsZ ring formation) (Chauhan et al, 2006; Dziadek et al, 2002Dziadek et al, , 2003Huang et al, 2007;Rajagopalan et al, 2005) are relatively well studied in mycobacteria, nothing is known about the segregation of chromosomes in these bacteria.…”

“…Knowledge regarding the steps of the mycobacterial cell cycle (replication, chromosome segregation and cell division) seems to be critical for understanding the mechanisms that are responsible for the transition from an active to a non-replicative persistent state (and vice versa) of pathogenic mycobacteria, particularly M. tuberculosis. While initiation of chromosome replication Qin et al, 1999;Rajagopalan et al, 1995;Zawilak et al, 2004) and cell division (FtsZ ring formation) (Chauhan et al, 2006; Dziadek et al, 2002Dziadek et al, , 2003Huang et al, 2007;Rajagopalan et al, 2005) are relatively well studied in mycobacteria, nothing is known about the segregation of chromosomes in these bacteria.Bacterial chromosome segregation has been recently found to be an active and complex process closely coupled with replication (see Bartosik & Jagura-Burdzy, 2005;Errington et al, 2005;Hayes & Barilla, 2006; Leonard et al, 2005 for reviews). In bacteria studied to date, the newly synthesized origin (oriC) regions undergo a symmetric or asymmetric segregation process; two copies of the duplicated oriC regions migrate from the cell centre toward opposite cell poles, i.e.…”

“…All the PCR-derived clones were analysed by DNA sequencing to check their fidelity. The fusion proteins, glutathione S-transferase (GST)-ParA or 6His-ParB were purified on a glutathione Sepharose 4B or on a Ni 2+ -NTA-agarose column (Qiagen), respectively, as described previously (Majka et al, 1999;Zawilak et al, 2004). For removal of the GST part, the bound fusion proteins were treated with the PreScission protease (Amersham-Pharmacia Biotech) and the ParA proteins were released from the beads.…”