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.
Highlights
Single-molecule imaging and manipulation approaches have greatly expanded our understanding of protein-nucleic acid interactions[1]
Our results show that yPCNA can be loaded on a 5′-single stranded DNA flap, a (CAG)[13] triplet nucleotide repeat (TNR), and homoduplex DNA
We sought to develop a method that fulfilled three criteria: (i) multiple exogenous DNA sequences can be inserted into λ-phage DNA with nearly 100% efficiency, (ii) the insertion positions are not limited by availability of unique restriction sites, and (iii) chemical modifications and extra-helical structures can be introduced efficiently and with minimal handling
Summary
Single-molecule imaging and manipulation approaches have greatly expanded our understanding of protein-nucleic acid interactions[1]. Introducing site-specific synthetic oligonucleotides and extrahelical structures poses additional challenges in such a long DNA substrate Such structures are usually inserted via a nicking endonuclease (nickase) based strategy[12, 16,17,18,19]. Nickase-based insertion is inefficient for wild type λ-DNA because nickases that produce closely-spaced nicks cleave hundreds of additional sites on both the top and bottom DNA strands, fragmenting the DNA substrate and reducing the overall yield of full-length DNA molecules Because this strategy relies on naturally occurring nick sites, it precludes site-specific incorporation of extrahelical structures at user-specified positions along the DNA substrate. We use in vivo recombineering to target any segment of a lysogenic phage, abrogating the need for restriction sites and ligation Using this approach, we develop a molecular toolkit for inserting exogenous DNA sequences into the λ-phage genome with >90% efficiency. We anticipate that this molecular toolkit will be broadly useful for both ensemble and single-molecule studies that require site-specific modification of long DNA substrates
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