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Methods Enzymol. 2017;582:221-238. doi: 10.1016/bs.mie.2016.08.006. Epub 2016 Oct 24.

Inserting Extrahelical Structures into Long DNA Substrates for Single-Molecule Studies of DNA Mismatch Repair.

Author information

1
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, United States.
2
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, United States; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX, United States. Electronic address: ifinkelstein@cm.utexas.edu.

Abstract

The DNA mismatch repair (MMR) system corrects errors that occur during DNA replication. MMR needs the coordinated and highly dynamic assembly of repair enzymes at the site of the lesion. By visualizing transient intermediates of these assemblies, single-molecule approaches have shed critical insights into the mechanisms of MMR. These studies frequently require long (>20kb) DNA substrates with lesions and other extrahelical structures inserted at defined positions. DNA derived from bacteriophage λ (λ-DNA) is a high quality long (48.5kb) DNA substrate that is frequently used in single-molecule studies. Here we provide detailed protocols for site-specific incorporation of recombinant sequences and extrahelical structures into λ-DNA. We also describe how to assemble DNA curtains, and how to collect and analyze single-molecule observations of lesion recognition by MMR proteins diffusing on these DNA curtains. These protocols will facilitate future single-molecule studies of DNA transcription, replication, and repair.

KEYWORDS:

DNA curtains; Nicking enzyme; Recombineering; Single-molecule imaging

PMID:
28062036
DOI:
10.1016/bs.mie.2016.08.006
[Indexed for MEDLINE]

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