Affinity and Sequence Specificity of DNA Binding and Site Selection for Primer Synthesis by <i>Escherichia coli</i> Primase

Khopde, Sujata M.; Biswas, Esther E.; Biswas, Subhasis B.

doi:10.1021/bi026711m

Cited by 32 publications

(46 citation statements)

References 35 publications

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“…Our previous studies have indicated that, in the absence of DnaB helicase, primase binds to DNA in a sequential dimer pathway without forming a true dimer (41). We observed a very negligible change in the anisotropy when the primase was added to Ru[bipy] 3 -primase, which supported either a very weak or no direct interaction between primase monomers.…”

Section: Analysis Of In Vitro Primer Synthesis Carried Out By Primasesupporting

confidence: 53%

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Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

Mitkova

Khopde

Biswas

2003

Journal of Biological Chemistry

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Initiation and synthesis of RNA primers in the lagging strand of the replication fork in Escherichia coli requires the replicative DnaB helicase and the DNA primase, the DnaG gene product. In addition, the physical interaction between these two replication enzymes appears to play a role in the initiation of chromosomal DNA replication. In vitro, DnaB helicase stimulates primase to synthesize primers on single-stranded (ss) oligonucleotide templates. Earlier studies hypothesized that multiple primase molecules interact with each DnaB hexamer and single-stranded DNA. We have examined this hypothesis and determined the exact stoichiometry of primase to DnaB hexamer. We have also demonstrated that ssDNA binding activity of the DnaB helicase is necessary for directing the primase to the initiator trinucleotide and synthesis of 11-20-nucleotide long primers. Although, association of these two enzymes determines the extent and rate of synthesis of the RNA primers in vitro, direct evidence of the formation of primase-DnaB complex has remained elusive in E. coli due to the transient nature of their interaction. Therefore, we stabilized this complex using a chemical crosslinker and carried out a stoichiometric analysis of this complex by gel filtration. This allowed us to demonstrate that the primase-helicase complex of E. coli is comprised of three molecules of primase bound to one DnaB hexamer. Fluorescence anisotropy studies of the interaction of DnaB with primase, labeled with the fluorescent probe Ru(bipy) 3 , and Scatchard analysis further supported this conclusion. The addition of DnaC protein, leading to the formation of the DnaB-DnaC complex, to the simple priming system resulted in the synthesis of shorter primers. Therefore, interactions of the DnaB-primase complex with other replication factors might be critical for determining the physiological length of the RNA primers in vivo and the overall kinetics of primer synthesis.During the last few decades, studies on the replication of phage, plasmid, and chromosomal DNA in Escherichia coli and eukaryotic cells have established an understanding of some of the basic mechanisms of DNA replication (1-4). Reconstitution of DNA replication with purified proteins has yielded great insight into the mechanism of DNA replication as well as other aspects of DNA metabolism, such as DNA repair and recombination in prokaryotic and eukaryotic cells (5-10). The replication of the E. coli chromosome requires a large number of proteins that have to work in concert in order to successfully accomplish initiation, elongation, and termination of DNA replication (2,(11)(12)(13)(14). Thus, a careful analysis of the interactions between replication factors is of critical importance for gaining further insights into the mechanism and control of the major steps of DNA replication.Upon DnaA protein activation of the origin, DnaB helicase enters the partially unwound origin. Binding of DnaB to singlestranded DNA (ssDNA) 1 is controlled by its interaction with DnaC. Association with DnaB sti...

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Section: Analysis Of In Vitro Primer Synthesis Carried Out By Primasesupporting

confidence: 53%

“…Our previous studies on primase-ssDNA binding demonstrated that it associates with DNA as a pseudo dimer (41). However, these studies were carried out in the absence of DnaB and did not preclude the possible formation of a higher order complex.…”

Section: Stoichiometry Of the Complex [Primase-dnab Hexamer] Ismentioning

confidence: 99%

Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

Mitkova

Khopde

Biswas

2003

Journal of Biological Chemistry

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“…In addition, DnaB also modulates primer synthesis in a manner independent of its translocation ability. It relaxes the specificity of the primase, stimulates its activity, and reduces the length of primers to between 10 and 14 nucleotides (1,7,8,10,24). Given the striking differences in the stability of the B. stearothermophilus DnaB-DnaG complex, it is not clear whether these observations apply only to E. coli.…”

mentioning

confidence: 99%

In the Bacillus stearothermophilus DnaB-DnaG Complex, the Activities of the Two Proteins Are Modulated by Distinct but Overlapping Networks of Residues

Thirlway

Soultanas

2006

J Bacteriol

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We demonstrate the primase activity of Bacillus stearothermophilus DnaG and show that it initiates at 3-ATC-5 and 3-ATT-5 sites synthesizing primers that are 22 or 23 nucleotides long. In the presence of the helicase DnaB the size distribution of primers is different, and a range of additional smaller primers are also synthesized. Nine residues from the N-and C-terminal domains of DnaB, as well as its linker region, have been reported previously to affect this interaction. In Bacillus stearothermophilus only three residues from the linker region (I119 and I125) and the N-terminal domain (Y88) of DnaB have been shown previously to have direct structural importance, and I119 and I125 mediate DnaG-induced effects on DnaB activity. The functions of the other residues (L138, T191, E192, R195, and M196) are still a mystery. Here we show that the E15A, Y88A, and E15A Y88A mutants bind DnaG but are not able to modulate primer size, whereas the R195A M196A mutant inhibited the primase activity. Therefore, four of these residues, E15 and Y88 (N-terminal domain) and R195 and M196 (C-terminal domain), mediate DnaB-induced effects on DnaG activity. Overall, the data suggest that the effects of DnaB on DnaG activity and vice versa are mediated by distinct but overlapping networks of residues.

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“…The E. coli DnaG gene product is the model eubacterial primase because its structure and function have been extensively characterized. It has been demonstrated that the E. coli primase is slow (9), has low-fidelity (9), binds G4-ori ssDNA as a dimer (10,11), and that DnaB helicase stimulates its catalytic activity over 15-fold (12 Recently, it was demonstrated that the primases and replication fork helicases from mesophilic S. aureus and thermophilic Geobacillus stearothermophilus have properties that diverge from their E. coli homologs (14,15). Both primases initiated from 5'-TTA-3' and 5'-CTA-3'.…”

Section: Staphylococcus Aureusmentioning

confidence: 99%

New Therapeutic Strategies for Antibiotic-Resistant Select Agents

Hinrichs¹,

Griep²

2007

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The public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.Send comments regarding this burden estimate or any other aspect of this collection of information, including suggesstions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA, 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any oenalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. a. REPORTNew Therapeutic Strategies for Antibiotic-Resistant Select Agents 14. ABSTRACT SECURITY CLASSIFICATION OF:The goal of this project was to establish the scientific basis for a new class of antibiotics that are needed to combat the development of resistance to currently available medications or the engineering of resistance into biological select agents. A multidisciplinary team of microbiologists, biochemists, molecular biologists and drug discovery experts was established to explore the hypothesis that disruption of bacterial primase activity will provide the basis for antimicrobial discovery and further, that the modular components of primase will provide for the discovery of broad-and narrow-spectrum antibiotics. The experimental model examined the functional activities of the Gram positive Staphylococcus aureus with the Gram negative E. S REPORT DATE (DD-MM-YYYY) TITLE AND SUBTITLE 31-12-200713. SUPPLEMENTARY NOTES The views, opinions and/or findings contained in this report are those of the author(s) and should not contrued as an official Department of the Army position, policy or decision, unless so designated by other documentation. Approved for public release; Federal purpose rights UL New Therapeutic Strategies for Antibiotic-Resistant Select AgentsReport Title ABSTRACTThe goal of this project was to establish the scientific basis for a new class of antibiotics that are needed to combat the development of resistance to currently available medications or the engineering of resistance into biological select agents. A multidisciplinary team of microbiologists, biochemists, molecular biologists and drug discovery experts was established to explore the hypothesis that disruption of bacterial primase activity will provide the basis for antimicrobial discovery and further, that the modular components of primase will provide for the discovery of broad-and narrow-spectrum antibiotics. The experimental model examined the functional activities of the Gram positive Staphylococcus aureus with the Gram negative E. coli for purposes of establishing a protocol for extension to select agents including Bacillus anthracis and Yersinia pestis. Impo...

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Affinity and Sequence Specificity of DNA Binding and Site Selection for Primer Synthesis by Escherichia coli Primase

Cited by 32 publications

References 35 publications

Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

In the Bacillus stearothermophilus DnaB-DnaG Complex, the Activities of the Two Proteins Are Modulated by Distinct but Overlapping Networks of Residues

New Therapeutic Strategies for Antibiotic-Resistant Select Agents

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