Format

Send to

Choose Destination
DNA Repair (Amst). 2014 Aug;20:94-109. doi: 10.1016/j.dnarep.2014.02.004. Epub 2014 Mar 2.

Visualizing protein movement on DNA at the single-molecule level using DNA curtains.

Author information

1
Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA.
2
Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA; Howard Hughes Medical Institute, USA. Electronic address: ecg2108@columbia.edu.

Abstract

A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively.

KEYWORDS:

Chromosome segregation; DNA curtains; Diffusion; FtsK; Homologous recombination; Mismatch repair; RecBCD; Translocation

PMID:
24598576
PMCID:
PMC4111998
DOI:
10.1016/j.dnarep.2014.02.004
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center