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Force-activated DNA substrates for probing individual proteins interacting with single-stranded DNA. Public Deposited

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https://scholar.colorado.edu/concern/articles/st74cr15s
Abstract
  • Single-molecule force spectroscopy provides insight into how proteins bind to and move along DNA. Such studies often embed a single-stranded (ss) DNA region within a longer double-stranded (ds) DNA molecule. Yet, producing these substrates remains laborious and inefficient, particularly when using the traditional three-way hybridization. Here, we developed a force-activated substrate that yields an internal 1000 nucleotide (nt) ssDNA region when pulled partially into the overstretching transition (∼65 pN) by engineering a 50%-GC segment to have no adjacent GC base pairs. Once the template was made, these substrates were efficiently prepared by polymerase chain reaction amplification followed by site-specific nicking. We also generated a more complex structure used in high-resolution helicase studies, a DNA hairpin adjacent to 33 nt of ssDNA. The temporally defined generation of individual hairpin substrates in the presence of RecQ helicase and saturating adenine triphosphate let us deduce that RecQ binds to ssDNA via a near diffusion-limited reaction. More broadly, these substrates enable the precise initiation of an important class of protein-DNA interactions.
Creator
Date Issued
  • 2017-10-13
Academic Affiliation
Journal Title
Journal Issue/Number
  • 18
Journal Volume
  • 45
File Extent
  • 10775-10782
Subject
Publisher
Last Modified
  • 2019-12-05
Identifier
  • PubMed ID: 28977580
Resource Type
Rights Statement
DOI
ISSN
  • 1362-4962
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