Efficient modification of λ-DNA substrates for single-molecule studies

Scientific Reports
Yoori KimI J Finkelstein

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 th...Continue Reading

References

Mar 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·A SimonsB Müller-Hill
Jun 1, 1995·Journal of Bacteriology·C Y ChangR Young
Mar 26, 1998·Journal of Biomolecular Structure & Dynamics·S V MariappanG Gupta
May 20, 1998·Proceedings of the National Academy of Sciences of the United States of America·H M Thorpe, M C Smith
Jun 1, 2000·Proceedings of the National Academy of Sciences of the United States of America·K A Datsenko, B L Wanner
Jun 18, 2004·Nature·Gregory D BowmanJohn Kuriyan
Jun 24, 2004·Progress in Nucleic Acid Research and Molecular Biology·Jerzy Majka, Peter M J Burgers
Jul 26, 2005·Nucleic Acids Research·Samir AmraneJean-Louis Mergny
Jun 4, 2008·Annual Review of Biochemistry·Carlos Bustamante
Jan 31, 2009·Nature Protocols·Shyam K SharanDonald L Court
May 5, 2009·The Journal of Biological Chemistry·Anna B KochaniakAntoine M van Oijen
Nov 13, 2010·Nucleic Acids Research·Nicholas LuzziettiRalf Seidel
Dec 15, 2010·Biochemistry·Natalya N DegtyarevaJeffrey T Petty
Jan 20, 2011·Proceedings of the National Academy of Sciences of the United States of America·Joseph J LoparoAntoine M van Oijen
Mar 19, 2011·Physical Chemistry Chemical Physics : PCCP·Andrea CandelliErwin J G Peterman
Jun 11, 2011·Methods in Molecular Biology·Ilya J Finkelstein, Eric C Greene
Mar 10, 2012·Nature Protocols·Nicholas LuzziettiRalf Seidel
Jan 1, 2006·CSH Protocols·Joseph Sambrook, David W Russell
Jun 30, 2012·Nature Methods·Johannes SchindelinAlbert Cardona
Aug 25, 2012·Sub-cellular Biochemistry·Lynne M DieckmanM Todd Washington
Sep 27, 2012·Proceedings of the National Academy of Sciences of the United States of America·Jason GormanEric C Greene
Oct 30, 2012·Nature·Jason C BellStephen C Kowalczykowski
Dec 4, 2012·Nature·Hasan YardimciJohannes C Walter
Jul 11, 2013·Proceedings of the National Academy of Sciences of the United States of America·Anna PluciennikPaul Modrich
Apr 17, 2014·Analytical Chemistry·Aaron D Robison, Ilya J Finkelstein
Aug 28, 2014·Nucleic Acids Research·John P Berezney, Omar A Saleh
Sep 26, 2014·Current Opinion in Structural Biology·Juan Carlos CordovaMatthew J Lang
Sep 30, 2014·Nature Methods·Taekjip Ha
Jan 13, 2015·Annual Review of Biochemistry·Ravi R IyerRobert D Wells
Apr 10, 2015·ACS Synthetic Biology·Gur PinesRyan T Gill

❮ Previous
Next ❯

Citations

Feb 13, 2019·BioTechniques·Samuel Jw KrerowiczDavid C Schwartz
Oct 4, 2017·BMC Biology·Alexandra SisakovaLumir Krejci
Oct 31, 2019·Cell Reports·Anna L MallamKevin Drew

❮ Previous
Next ❯

Methods Mentioned

BETA
fluorescence
FRET
fluorescence microscopy
PCR
electrophoresis
gel filtration
restriction digest
fluorescence imaging

Software Mentioned

NIS
PAST3
MATLAB
Elements
FIJI
ImageJ

Related Concepts

Related Feeds

Bacteriophage: Phage Therapy

Phage therapy uses bacterial viruses (bacteriophages) to treat bacterial infections and is widely being recognized as an alternative to antibiotics. Here is the latest research.

Related Papers

Current Opinion in Structural Biology
S E Phillips, D Moras
Acta Biochimica Et Biophysica; Academiae Scientiarum Hungaricae
C Hélène
Current Opinion in Structural Biology
© 2022 Meta ULC. All rights reserved