The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

Jeong, Young Jin; Levin, Mikhail K.; Patel, Smita S.

doi:10.1073/pnas.0400372101

Cited by 88 publications

(105 citation statements)

References 47 publications

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“…On the other hand, an ensemble study of the ring-shaped helicase (E. coli DnaB) indicated an almost 7-fold difference in the unwinding rate depending on the sequence of the dsDNA substrate (39). Similarly, ensemble studies have shown that the non-ring-shaped UvrD helicase and ring-shaped T7 helicase, respectively, move 3-9-fold faster on ssDNA relative to their rates of dsDNA unwinding (29,40,41). 4 The latter observations imply that the unwinding rates of these helicases might depend on base pair stability.…”

mentioning

confidence: 95%

“…The length of the dsNA region of the substrates was chosen carefully; it cannot be too short, because at room temperature 8 -16 bp of dsNA of a particular sequence can have ⌬G close to zero and can get spontaneously unwound (29,42). The length of the dsNA region cannot be too long, otherwise the unwound strands may reanneal behind the helicase during the reaction (29).…”

Section: Methodsmentioning

confidence: 99%

“…Experimental data with only two states was fit to the Equation 1 (29,50) to obtain the rate and processivity parameters,…”

Section: Methodsmentioning

confidence: 99%

“…T7 helicase is an essential component of the replisome that consists of T7 DNA polymerase, and singlestranded binding protein from the host and from the phage (24 -27). It has been shown that T7 helicase uses the energy from dTTP hydrolysis to translocate unidirectionally along ssDNA 3 and to unwind the strands of dsDNA (28,29). A member of the non-ring-shaped helicases that we have studied here is encoded by the hepatitis C virus (HCV).…”

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confidence: 99%

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Nucleic Acid Unwinding by Hepatitis C Virus and Bacteriophage T7 Helicases Is Sensitive to Base Pair Stability

Donmez¹,

Rajagopal²,

Jeong

et al. 2007

Journal of Biological Chemistry

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Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.Helicases are proteins that translocate in one specific direction along the nucleic acid backbone to catalyze processes such as the separation of the complementary strands of a double helical nucleic acid, recombination of DNA molecules, or remodeling of proteins bound to nucleic acids (1-5). Helicases are referred to as motor enzymes, because they convert the chemical energy of NTP hydrolysis (a reaction with a negative ⌬G) into mechanical force for motion and strand separation. It is not clear whether the force they generate is used predominantly to translocate on the nucleic acid or whether it is directed mainly at actively destabilizing and melting the double-stranded nucleic acid. It is also not known whether all helicases share a common mechanism or whether they act via different strategies to catalyze strand separation. Therefore, it is difficult to predict a priori whether and how the helicase activity would depend on nucleic acid base pair stability.Helicases encounter a range of nucleic acid sequences as they translocate on and unwind the strands of double-stranded DNA or RNA in the cell. It is well known that the base sequence dictates the stability of the double helical nucleic acid. The GC base pair in nucleic acids is more stable than the AT base pair. Similarly, the neighboring base sequences and the stacking energies between adjacent base pairs determine the overall stability of the double helical nucleic acid (6). A strong dependence of helicase activity on base pair stability would adversely affect controlled biological processes, such as the coordination of leading and lagging strand DNA synthesis during genome replication or the repair and recombination of specific DNA sequences. On the other hand, the dependence of helicase activity on base pair stability pr...

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mentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

“…Experimental data with only two states was fit to the Equation 1 (29,50) to obtain the rate and processivity parameters,…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Nucleic Acid Unwinding by Hepatitis C Virus and Bacteriophage T7 Helicases Is Sensitive to Base Pair Stability

Donmez¹,

Rajagopal²,

Jeong

et al. 2007

Journal of Biological Chemistry

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“…Indeed, the biochemical and structural studies have shown that some helicases assemble into stable cooperative hexameric rings (3). These helicases include the Escherichia coli DnaB (6, 7) and Rho (8, 9), bacteriophage T4 gp41 (10, 11), and T7 gp4 (12)(13)(14). DNA passes through such a central channel and is unwound with a high processivity.…”

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confidence: 99%

Escherichia coli RecQ Is a Rapid, Efficient, and Monomeric Helicase

Zhang

Dou

Xie

et al. 2006

Journal of Biological Chemistry

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RecQ family helicases play a key role in chromosome maintenance. Despite extensive biochemical, biophysical, and structural studies, the mechanism by which helicase unwinds double-stranded DNA remains to be elucidated. Using a wide array of biochemical and biophysical approaches, we have previously shown that the Escherichia coli RecQ helicase functions as a monomer. In this study, we have further characterized the kinetic mechanism of the RecQ-catalyzed unwinding of duplex DNA using the fluorometric stopped-flow method based on fluorescence resonance energy transfer. Our results show that RecQ helicase binds preferentially to 3-flanking duplex DNA. Under the pre-steady-state conditions, the burst amplitude reveals a 1:1 ratio between RecQ and DNA substrate, suggesting that an active monomeric form of RecQ helicase is involved in the catalysis. Under the single-turnover conditions, the RecQ-catalyzed unwinding is independent of the 3-tail length, indicating that functional interactions between RecQ molecules are not implicated in the DNA unwinding. It was further determined that RecQ unwinds DNA rapidly with a step size of 4 bp and a rate of ϳ21 steps/s. These kinetic results not only further support our previous conclusion that E. coli RecQ functions as a monomer but also suggest that some of the Superfamily 2 helicases may function through an "inchworm" mechanism.Helicases are molecular motor proteins that use the energy of nucleotide triphosphate hydrolysis to translocate along and separate the complementary strands of a nucleic acid duplex. These enzymes play essential roles in most aspects of the DNA metabolic pathway, such as replication, repair, recombination, and transcription (1-5). A large number of helicases have been identified; however, the mechanisms by which helicases unwind double-stranded DNA (dsDNA) 3 remain obscure.One of the major concerns in studying the unwinding mechanism of a DNA helicase is its oligomeric state. This is because an oligomeric structure could provide multiple potential binding sites for the DNA substrate and nucleotide cofactors, which are absolutely required for the helicase to translocate along the DNA track during the processive DNA unwinding. Indeed, the biochemical and structural studies have shown that some helicases assemble into stable cooperative hexameric rings (3). These helicases include the Escherichia coli DnaB (6, 7) and Rho (8, 9), bacteriophage T4 gp41 (10, 11), and T7 gp4 (12)(13)(14). DNA passes through such a central channel and is unwound with a high processivity. For the non-ring helicases, the enzymes could be assembled into oligomers, and the DNA binding sites may be located on the separate subunits. On the basis of quantitative analyses of DNA binding properties, a "rolling model" was proposed to explain factorial DNA unwinding (15). This model suggests that each monomer of the Rep dimer binds alternatively to ssDNA and dsDNA. This process is regulated by repeated binding and hydrolysis of ATP and release of ADP. In this way, Rep dimer rolls along ...

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