Efficient modification of λ-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 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.

Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex

“…PCR products containing the large homology arms were recombineered into E. coli lysogens and the recombinant DNA purified from packaged phage particles(Kim et al, 2017). To functionalize the DNA ends for single-molecule experiments, we combine 125 µg of purified λ-phage DNA with 2 µM of biotinylated oligos (IF001or IF003 or (Table S1)).…”

Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex

“…Single-molecule nicking assay: 5 nM PCNA was loaded by 1.5 nM RFC on double-tethered DNA curtain in Mlh1-Pms1 endonuclease buffer (40 mM Tris-HCl pH 8.0, 0.2 mg mL -1 BSA, 50 mM NaCl, 2 mM MnCl2, 1 mM DTT, 1 mM ATP) (54). MgCl2 was used instead of MnCl2 for manganese negative control.…”

“…This reaction was carried out in two steps. First, PCNA was loaded by RFC on doubletethered DNA curtains in the presence of ATP, as described previously (54). After flushing out RFC, Mlh1-Pms1 was incubated in the flowcell for 20 minutes (Figure 4D).…”

Systematic discovery of endogenous human ribonucleoprotein complexes

“…RFC was purified as previously described (Finkelstein et al, 2003;Kim et al, 2017). Briefly, full-length S. cerevisiae RFC was expressed in BL21(DE3) ArcticExpress (Agilent) E. coli cotransformed with pLant2b-RFC-AE (pIF117) and pET11-RFC-BCD (pIF116).…”

“…Prior to protein identification, human and mouse proteomes were downloaded from UniProt website (Apweiler et al, 2004). Raw formatted mass spectrometry files were first converted to mzXML file format using MSConvert (http://proteowizard.sourceforge.net/tools.shtml) and then processed using MSGF+ (Kim et al, 2017), X! TANDEM (Craig and Beavis, 2004) and…”