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Processivity, Velocity, and Universal Characteristics of Nucleic Acid Unwinding by Helicases.

ABSTRACT: Helicases are components of the cellular replisome that are essential for unwinding double-strand nucleic acids during the process of replication. Intriguingly, most helicases are inefficient and require either oligomerization or assistance from other partner proteins to increase the processivity of unwinding in the presence of the replication fork, which acts as a barrier to progress. Single-molecule force spectroscopy has emerged as a promising experimental technique to probe how relieving this barrier on the helicase can allow for increased efficiency of unwinding. However, there exists no comprehensive theoretical framework to provide unique interpretations of the underlying helicase kinetics from the force spectroscopy data. This remains a major confounding issue in the field. Here, we develop a mathematical framework and derive analytic expressions for the velocity and run length of a general model of finitely processive helicases, the two most commonly measured experimental quantities. We show that in contrast to the unwinding velocity, the processivity exhibits a universal increase in response to external force, irrespective of the underlying architecture and unwinding kinetics of the helicase. Our work provides the first, to our knowledge, explanation to a wide array of experiments and suggests that helicases may have evolved to maximize processivity rather than speed. To demonstrate the use of our theory on experimental data, we analyze velocity and processivity data on the T7 helicase and provide unique inferences on the kinetics of the helicase. Our results show that T7 is a weakly active helicase that destabilizes the fork ahead by less than 1 kBT and back steps very frequently while unwinding DNA. Our work generates fundamental insights into the force response of helicases and provides a widely applicable method for inferring the underlying helicase kinetics from force spectroscopy data.

SUBMITTER: Chakrabarti S

PROVIDER: S-EPMC6731385 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Processivity, Velocity, and Universal Characteristics of Nucleic Acid Unwinding by Helicases.

Chakrabarti Shaon S Jarzynski Christopher C Thirumalai D D

Biophysical journal 20190720 5

Helicases are components of the cellular replisome that are essential for unwinding double-strand nucleic acids during the process of replication. Intriguingly, most helicases are inefficient and require either oligomerization or assistance from other partner proteins to increase the processivity of unwinding in the presence of the replication fork, which acts as a barrier to progress. Single-molecule force spectroscopy has emerged as a promising experimental technique to probe how relieving thi ...[more]

PMID: 31400912

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Project description:BackgroundMultiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping.ResultsWe designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method.ConclusionAs a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.

Dataset Information

Processivity, Velocity, and Universal Characteristics of Nucleic Acid Unwinding by Helicases.

Publications

Processivity, Velocity, and Universal Characteristics of Nucleic Acid Unwinding by Helicases.

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