Bacillus anthracisandBacillus cereusPcrA Helicases Can Support DNA Unwinding and In Vitro Rolling-Circle Replication of Plasmid pT181 ofStaphylococcus aureus

Anand, Syam P.; Mitra, Poulami; Naqvi, Asma; Khan, Saleem A.

doi:10.1128/jb.186.7.2195-2199.2004

Cited by 32 publications

(32 citation statements)

References 37 publications

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“…The failure to establish pT181 in S. pneumoniae suggested that this staphylococcal plasmid was unable to replicate in this bacterium, in agreement with the apparent inability of the RepCPcrA Spn complex to efficiently unwind the pT181 DNA. In contrast to these results, heterologous PcrA helicases from Bacillus cereus PcrA Bcer and Bacillus anthracis PcrA Ban (two species in which the pT181 replicon could be established) are known to extensively unwind RepC-nicked pT181 DNA and support pT181 replication in vitro (3). We propose that a functional interaction between the plasmid-encoded replication initiator protein and the host PcrA helicase is essential for establishment and replication of RCR plasmids in gram-positive bacteria.…”

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confidence: 42%

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Genetic and Biochemical Characterization of the Streptococcus pneumoniae PcrA Helicase and Its Role in Plasmid Rolling Circle Replication

et al. 2006

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PcrA is a chromosomally encoded DNA helicase of gram-positive bacteria involved in replication of rolling circle replicating plasmids. Efficient interaction between PcrA and the plasmid-encoded replication initiator (Rep) protein is considered a requirement for the plasmid to replicate in a given host, and thus, the ability of a Rep protein to interact with heterologous PcrA helicases has been invoked as a determinant of plasmid promiscuity. We characterized transcription of the Streptococcus pneumoniae pcrA gene in its genetic context and studied the biochemical properties of its product, the PcrA Spn helicase. Transcription of the pneumococcal pcrA gene was directed by promoter Pa, consisting of an extended ؊10 box. Promoter Pa also accounted for expression of a second essential gene, radC, which was transcribed with much lower efficiency than pcrA, probably due to the presence of a terminator/attenuator sequence located between the two genes. PcrA Spn displayed single-stranded DNA-dependent ATPase activity. PcrA Spn showed 533 and 335 helicase activities and bound efficiently to partially duplex DNA containing a hairpin structure adjacent to a 6-nucleotide 5 or 3 single-stranded tail and one unpaired (flap) nucleotide in the complementary strand. PcrA Spn interacted specifically with RepC, the initiator of staphylococcal plasmid pT181. Although the pneumococcal helicase was able to initiate unwinding of the RepC-nicked pT181 DNA, it was much less processive in this activity than the cognate staphylococcal PcrA protein. Accordingly, PcrA Spn was inefficient in in vitro replication of pT181, and perhaps as a consequence, this plasmid could not be established in S. pneumoniae.

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confidence: 42%

“…The transcriptional terminator of the cat gene and the transcriptional terminator proposed to attenuate transcription of radC are indicated by hairpin symbols. (3,9,34), the ATPase activity of His-PcrA Spn was significantly stimulated by addition of ssDNA to the reaction mixtures (Fig. 4).…”

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confidence: 99%

Genetic and Biochemical Characterization of the Streptococcus pneumoniae PcrA Helicase and Its Role in Plasmid Rolling Circle Replication

et al. 2006

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“…poly(dT) 4 3.06 Ϯ 0.65 23.3 Ϯ 0.2 poly(dT) 6 1.99 Ϯ 0.38 16.4 Ϯ 0.1 poly(dT) 10 0.37 Ϯ 0.03 32.7 Ϯ 0.4 poly(dT) 13 0.16 Ϯ 0.03 27.9 Ϯ 0.1 poly(dT) 18 0.059 Ϯ 0.003 29.4 Ϯ 0.3 poly(dT) 23 0.045 Ϯ 0.004 24.7 Ϯ 0.5 poly(dT) 40 0 (Fig. 7A) showed a nonsigmoidal increase in ATPase rate with the protein concentration in the range tested (20 nM to 500 nM), indicating the absence of any significant cooperativity between protein molecules.…”

Section: Resultsmentioning

confidence: 99%

“…B. stearothermophilus PcrA shows a strong preference for double-stranded substrates containing a 3Ј single-stranded tail over substrates containing a 5Ј singlestranded tail (19). Interestingly, Bacillus anthracis, Bacillus cereus, and S. aureus PcrAs were equally active as 5Ј-3Ј helicases and 3Ј-5Ј helicases (4,48).…”

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confidence: 99%

Characterization of the Helicase Activity and Substrate Specificity of Mycobacterium tuberculosis UvrD

Curti¹,

Smerdon²,

Davis³

2007

J Bacteriol

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UvrD is a helicase that is widely conserved in gram-negative bacteria. A uvrD homologue was identified in Mycobacterium tuberculosis on the basis of the homology of its encoded protein with Escherichia coli UvrD, with which it shares 39% amino acid identity, distributed throughout the protein. The gene was cloned, and a histidine-tagged form of the protein was expressed and purified to homogeneity. The purified protein had in vitro ATPase activity that was dependent upon the presence of DNA. Oligonucleotides as short as four nucleotides were sufficient to promote the ATPase activity. The DNA helicase activity of the enzyme was only fueled by ATP and dATP. UvrD preferentially unwound 3-single-stranded tailed duplex substrates over 5-single-stranded ones, indicating that the protein had a duplex-unwinding activity with 3-to-5 polarity. A 3 single-stranded DNA tail of 18 nucleotides was required for effective unwinding. By using a series of synthetic oligonucleotide substrates, we demonstrated that M. tuberculosis UvrD has an unwinding preference towards nicked DNA duplexes and stalled replication forks, representing the likely sites of action in vivo. The potential role of M. tuberculosis UvrD in maintenance of bacterial genomic integrity makes it a promising target for drug design against M. tuberculosis.

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“…In all these strains, the role of PcrA in RCR has been studied using the broad host range pT181 plasmid as a reference (31,174). A clear interaction of the helicase with the replication initiation protein was successfully observed by pull-down for all three PcrAs.…”

Section: Role Of Superfamily Group 1 Helicases and Their Interaction mentioning

confidence: 99%

Plasmid Rolling-Circle Replication

et al. 2015

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Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

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Bacillus anthracisandBacillus cereusPcrA Helicases Can Support DNA Unwinding and In Vitro Rolling-Circle Replication of Plasmid pT181 ofStaphylococcus aureus

Cited by 32 publications

References 37 publications

Genetic and Biochemical Characterization of the Streptococcus pneumoniae PcrA Helicase and Its Role in Plasmid Rolling Circle Replication

Genetic and Biochemical Characterization of the Streptococcus pneumoniae PcrA Helicase and Its Role in Plasmid Rolling Circle Replication

Characterization of the Helicase Activity and Substrate Specificity of Mycobacterium tuberculosis UvrD

Plasmid Rolling-Circle Replication

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