Mechanism of DnaB Helicase of <i>Escherichia</i> <i>coli</i>:  Structural Domains Involved in ATP Hydrolysis, DNA Binding, and Oligomerization

Biswas, Esther E.; Biswas, Subhasis B.

doi:10.1021/bi990048t

Cited by 52 publications

(95 citation statements)

References 32 publications

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“…Evidently, DnaBMut2 was able to provide the functions of the helicase such as translocation on ssDNA template required for successful stimulation of the primase activity. These results were probably a consequence of the fact that mutations R324A and R326A in the RSRARR DNA binding motif of the helicase did not abolish, but significantly reduced the DNA binding and the DNA-dependent ATPase and helicase activities (46). In contrast, DnaBMut1 failed to stimulate the primase activity of the general priming system (Fig.…”

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

confidence: 97%

“…Our previous studies suggested that DnaBMut1, with specific mutations R328A and R329A in the RSRARR DNA binding motif exhibit a significant decrease in DNA-dependent ATPase activity and a complete loss of the helicase activity, indicating the important roles of these residues in DNA binding and helicase activity (46). Besides specific mutations, R324A and R326A of DnaBMut2 led to a considerably attenuated DNA binding as well as reduced DNA-dependent ATPase and helicase activities (46). We further addressed the question of how the mutations in RSRARR DNA binding will interfere with the primer synthesis of the general priming system.…”

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

confidence: 99%

“…On the other hand, the DNA binding site restricted to the motif RSRARR and the leucine zipper motif that is responsible for DnaB dimerization are localized in the opposite C-terminal domain of the DnaB helicase (46). Our previous studies suggested that DnaBMut1, with specific mutations R328A and R329A in the RSRARR DNA binding motif exhibit a significant decrease in DNA-dependent ATPase activity and a complete loss of the helicase activity, indicating the important roles of these residues in DNA binding and helicase activity (46). Besides specific mutations, R324A and R326A of DnaBMut2 led to a considerably attenuated DNA binding as well as reduced DNA-dependent ATPase and helicase activities (46).…”

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

confidence: 99%

“…2B, lanes 2-6). Therefore, it appeared that mutations R328A and R329A in the RSRARR DNA binding motif of DnaB not only repress the DNA binding and the helicase activity (46), but also attenuate its ability to cooperate with the primase during in vitro primer synthesis.…”

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

confidence: 99%

“…2A). This result is due to the fact that mutations R324A and R326B do not abolish, but rather significantly reduce, DNA binding and DNA-dependent ATPase and helicase activity (46). It has been shown that the DNA binding motif, RSRARR, is located in the C-terminal domain of E. coli helicase (45), whereas the primase binding site is positioned in the N-terminal domain (35).…”

Section: Dnab Helicase Binding To Dna Is Necessary For Directing Thementioning

confidence: 99%

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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 Primasementioning

confidence: 97%

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

confidence: 99%

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

confidence: 99%

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

confidence: 99%

Section: Dnab Helicase Binding To Dna Is Necessary For Directing Thementioning

confidence: 99%

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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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Purification and Characterization

Tsai

2006

Biomacromolecules

207

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Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) from I. loihiensis saline's bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize the I. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45˚C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD + and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.

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The Mycobacterium avium-intracellulare Complex dnaB Locus and Protein Intein Splicing

Yamamoto

Low

Rutherford

et al. 2001

Biochemical and Biophysical Research Communications

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Mechanism of DnaB Helicase of Escherichia coli: Structural Domains Involved in ATP Hydrolysis, DNA Binding, and Oligomerization

Cited by 52 publications

References 32 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

Purification and Characterization

The Mycobacterium avium-intracellulare Complex dnaB Locus and Protein Intein Splicing

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