General Methods for Analysis of Sequential “n-step” Kinetic Mechanisms: Application to Single Turnover Kinetics of Helicase-Catalyzed DNA Unwinding

Lucius, Aaron L.; Maluf, Nasib K.; Fischer, Christopher J.; Lohman, Timothy M.

doi:10.1016/s0006-3495(03)74648-7

Cited by 136 publications

(221 citation statements)

References 38 publications

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“…2. This model is similar to the one used to analyze the UvrD helicase kinetics (22,39), with some modifications. The stepping model assumes that the helicase separates a particular length L of dsDNA in n steps; thus, the step size s is L͞n.…”

Section: Resultsmentioning

confidence: 99%

“…The reason for the decrease in the step size of T7 helicase from Ϸ9 to Ϸ6 bp when the dTTP concentration is decreased from 2 mM to 200 M is not obvious and requires an understanding of the meaning of the kinetic step size. The kinetic step size is defined as the average number of base pairs unwound between two successive rate-limiting steps in the helicase reaction (22,39). If the rate-limiting step of T7 helicase at low and high dTTP is different, then this might be the reason for the dependence of the kinetic step size on dTTP.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Jeong

Levin

Patel

2004

Proc. Natl. Acad. Sci. U.S.A.

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Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding. We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters. The comparison of the unwinding properties of T7 helicase with its translocation properties on singlestranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases. T7 helicase unwinds DNA with a rate of 15 bp͞s, which is 9-fold slower than the translocation speed along ssDNA. T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA. We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel. The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.H elicases are ubiquitous proteins that are involved in various DNA and RNA metabolic processes that require the separation of double-stranded (ds)DNA into single strands, the removal of secondary structures in RNA, or the dissociation of proteins from nucleic acids (1-4). To perform these functions, helicases use the chemical energy from NTP hydrolysis to drive the mechanical processes of translocation and nucleic acid strand separation. In this paper, we study the mechanism of DNA unwinding by bacteriophage T7 helicase that is involved in DNA replication.During replication, the helicase has to unwind a long stretch of DNA, and that requires the helicase to couple strandseparation activity to translocation. The mechanisms of these critical processes of the helicase reaction are largely unknown. It is becoming evident that helicases can move unidirectionally along nucleic acid and displace bonded moieties along their path without specifically interacting with these moieties (5-8). Thus, unidirectional translocation is a basic activity that helicases can perform without requiring interactions with the duplex DNA. Nucleic acid strand separation is a thermodynamically unfavorable process and it is made feasible by the binding of the helicase to the newly unwound strands. Numerous mechanisms of unwinding have been proposed (2-4, 9-13), but additional experimental data are needed to distinguish between these mechanisms. For the monomeric or dimeric helicases such as the Escherichia coli PcrA and Rep helicases (14-16), it has been proposed that unwinding occurs by an active mechani...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Jeong

Levin

Patel

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…True or intrinsic processivity (P Intr ) is determined by the dissociation probability (P d ) of the enzyme from the polymer. It has also been demonstrated that P Intr corresponds to the average number of catalytic acts performed before the dissociation of the enzyme from the polymer (38). P Intr can realize only on an ideal polymer where P d is independent of the position of the enzyme on polymer.…”

Section: Measurement Of Cellulase Processivity; Methodsmentioning

confidence: 99%

Processivity of Cellobiohydrolases Is Limited by the Substrate

Kurašin

Väljamaë

2011

Journal of Biological Chemistry

245

354

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Processive cellobiohydrolases (CBHs) are the key components of fungal cellulase systems. Despite the wealth of structural data confirming the processive mode of action, little quantitative information on the processivity of CBHs is available. Here, we developed a method for measuring cellulase processivity. Sensitive fluorescence detection of enzyme-generated insoluble reducing groups on cellulose after labeling with diaminopyridine enabled quantification of the number of reducing-end exo-mode and endo-mode initiations. Both CBHs TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium employed reducing-end exoand endo-mode initiation in parallel. Processivity values measured for TrCel7A and PcCel7D on cellulose hydrolysis were more than an order of magnitude lower than the values of intrinsic processivity that were found from the ratio of catalytic constant (k cat ) and dissociation rate constant (k off ). We propose that the length of the obstacle-free path available for a processive run on cellulose chain limits the processivity of CBHs on cellulose. TrCel7A and PcCel7D differed in their k off values, whereas the k cat values were similar. Furthermore, the k off values for endoglucanases (EGs) were much higher than the k off values for CBHs, whereas the k cat values for EGs and CBHs were within the same order of magnitude. These results suggest that the value of k off may be the primary target for the selection of cellulases.

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“…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%

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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General Methods for Analysis of Sequential “n-step” Kinetic Mechanisms: Application to Single Turnover Kinetics of Helicase-Catalyzed DNA Unwinding

Cited by 136 publications

References 38 publications

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

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

Processivity of Cellobiohydrolases Is Limited by the Substrate

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

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