Purification and characterization of the PcrA helicase of Bacillus anthracis - PubMed (original) (raw)

Purification and characterization of the PcrA helicase of Bacillus anthracis

Asma Naqvi et al. J Bacteriol. 2003 Nov.

Abstract

PcrA is an essential helicase in gram-positive bacteria, and a gene encoding this helicase has been identified in all such organisms whose genomes have been sequenced so far. The precise role of PcrA that makes it essential for cell growth is not known; however, PcrA does not appear to be necessary for chromosome replication. The pcrA gene was identified in the genome of Bacillus anthracis on the basis of its sequence homology to the corresponding genes of Bacillus subtilis and Staphylococcus aureus, with which it shares 76 and 72% similarity, respectively. The pcrA gene of B. anthracis was isolated by PCR amplification and cloning into Escherichia coli. The PcrA protein was overexpressed with a His6 fusion at its amino-terminal end. The purified His-PcrA protein showed ATPase activity that was stimulated in the presence of single-stranded (ss) DNA (ssDNA). Interestingly, PcrA showed robust 3'-->5' as well as 5'-->3' helicase activities, with substrates containing a duplex region and a 3' or 5' ss poly(dT) tail. PcrA also efficiently unwound oligonucleotides containing a duplex region and a 5' or 3' ss tail with the potential to form a secondary structure. DNA binding experiments showed that PcrA bound much more efficiently to oligonucleotides containing a duplex region and a 5' or 3' ss tail with a potential to form a secondary structure than to those with ssDNAs or duplex DNAs with ss poly(dT) tails. Our results suggest that specialized DNA structures and/or sequences represent natural substrates of PcrA in biochemical processes that are essential for the growth and survival of gram-positive organisms, including B. anthracis.

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Figures

FIG. 1.

Alignment of PcrA of B. anthracis (Ba), B. stearothermophilus (Bst), B. subtilis (Bs), and S. aureus (Sa) in the seven conserved helicase motifs. Amino acids that differ from those of B. anthracis PcrA are shaded and boxed. Numbers correspond to the amino acid positions of the B. anthracis PcrA.

FIG. 2.

SDS-PAGE analysis of the purified B. anthracis PcrA protein. U, lysates from uninduced cells; I, lysates from IPTG-induced cells overexpressing the His-PcrA protein; P, PcrA protein purified by nickel affinity column chromatography; M, protein molecular-weight standards (in kilodaltons). Smaller fragments likely correspond to breakdown products of PcrA.

FIG. 3.

ATPase activity of PcrA. (A) Products of [α-32P]dATP hydrolysis in the presence of increasing amounts of PcrA. (B) Stimulation of the dATPase activity of PcrA by a 53mer ss oligonucleotide (ssDNA). The products of [α-32P]dATP hydrolysis were analyzed by thin-layer chromatography.

FIG. 4.

Helicase activity of the PcrA protein. 32P-labeled substrates were incubated with the indicated amounts of PcrA, and the products were resolved by native polyacrylamide gel electrophoresis. The probes used are listed in Table 1 and correspond to duplex oligonucleotides (oligo) containing either a 5′ or 3′ ss region. Only one strand of the probe was labeled. “Complex” corresponds to a PcrA-DNA complex.

FIG. 5.

Binding of the PcrA helicase to various DNA substrates. The PcrA helicase (200 ng) was incubated with probes labeled at the 5′ end, and the DNA-protein complexes were resolved by electrophoresis on native 6% polyacrylamide gels. The probes used are indicated. ss top, top strand of oligonucleotide d; ss bottom, bottom strand of oligonucleotide c. P, free probe; C, PcrA-DNA complex.

FIG. 6.

Dose-dependent binding of PcrA to duplex DNA substrates containing 5′ or 3′ ss regions (oligonucleotide [oligo] c or oligonucleotide d, respectively). 32P-labeled oligonucleotides were incubated with the indicated amounts of PcrA, and the products were resolved by polyacrylamide gel electrophoresis. P, free probe; C, PcrA-DNA complex.

References

1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. - PubMed
1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, and K. Struhl. 1988. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y.
1. Bird, L. E., S. Subramanya, and D. B. Wigley. 1998. Helicases: a unifying structural theme? Curr. Opin. Struct. Biol. 8:14-18. - PubMed
1. Bruand, C., and S. D. Ehrlich. 2000. UvrD-dependent replication of rolling-circle plasmids in Escherichia coli. Mol. Microbiol. 35:204-210. - PubMed
1. Bruck, I., and M. O'Donnell. 2000. The DNA replication machine of a gram-positive organism. J. Biol. Chem. 275:28971-28983. - PubMed

Purification and characterization of the PcrA helicase of Bacillus anthracis - PubMed (original) (raw)