Active DNA unwinding dynamics during processive DNA replication

Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecule...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2012-05, Vol.109 (21), p.8115-8120
Main Authors: Morin, José A, Cao, Francisco J, Lázaro, José M, Arias-Gonzalez, J. Ricardo, Valpuesta, José M, Carrascosa, José L, Salas, Margarita, Ibarra, Borja
Format: Article
Language:English
Subjects:
Bacillus Phages - genetics
Bending
Biological Sciences
Data lines
DNA
DNA Helicases - genetics
DNA Helicases - metabolism
DNA polymerase
DNA replication
DNA Replication - genetics
DNA, Viral - chemistry
DNA, Viral - genetics
DNA-directed DNA polymerase
DNA-Directed DNA Polymerase - genetics
DNA-Directed DNA Polymerase - metabolism
Gene Expression Regulation, Viral - physiology
Globular star clusters
Kinetics
Molecular Motor Proteins - physiology
Molecules
Motors
Nucleic Acid Conformation
nucleotide sequences
Nucleotides
Stress, Mechanical
Tunnels
Viral Proteins - genetics
Viral Proteins - metabolism
Y intersections
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Summary:Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1204759109