Efficient Modification of λ-DNA Substrates for Single-Molecule Studies

Sci Rep. 2017 May 18;7(1):2071. doi: 10.1038/s41598-017-01984-x.

Abstract

Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage λ is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into the λ-phage genome with 90-100% yield. Extrahelical structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of S. cerevisiae Proliferating Cell Nuclear Antigen (yPCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that S. cerevisiae Replication Factor C (yRFC) can load yPCNA onto 5'-ssDNA flaps, (CAG)13 triplet repeats, and homoduplex DNA. However, yPCNA remains trapped on the (CAG)13 structure, confirming a proposed mechanism for triplet repeat expansion. We anticipate that this molecular toolbox will be broadly useful for other studies that require site-specific modification of long DNA substrates.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteriophage lambda / genetics
  • Bacteriophage lambda / metabolism
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism*
  • DNA, Viral / genetics
  • DNA, Viral / metabolism*
  • Proliferating Cell Nuclear Antigen / metabolism
  • Protein Binding
  • Replication Protein C / metabolism
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism
  • Single Molecule Imaging / methods*
  • Trinucleotide Repeats

Substances

  • DNA, Single-Stranded
  • DNA, Viral
  • POL30 protein, S cerevisiae
  • Proliferating Cell Nuclear Antigen
  • Saccharomyces cerevisiae Proteins
  • Replication Protein C