Efficient modification of ?-DNA substrates for single-molecule studies.

ABSTRACT: Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage ? is a convenient source of high quality long (48.5?kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifications into ?-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 ?-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into the ?-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 ?-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.

SUBMITTER: Kim Y 

PROVIDER: S-EPMC5437064 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature