Document Type
Article
Language
eng
Publication Date
7-14-2016
Publisher
Public Library of Science (PLoS)
Source Publication
PLoS One
Source ISSN
1932-6203
Abstract
Single-stranded DNA binding (SSB) proteins coordinate DNA replication, repair, and recombination and are critical for maintaining genomic integrity. SSB binds to single-stranded DNA (ssDNA) rapidly and with very high affinity making it a useful molecular tool to detect free ssDNA in solution. We have labeled SSB from Plasmodium falciparum (Pf-SSB) with the MDCC (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)-carbonyl)coumarin) fluorophore which yields a four-fold increase in fluorescence upon binding to ssDNA. Pf-SSBMDCC binding to DNA is unaffected by NaCl or Mg2+ concentration and does not display salt-dependent changes in DNA binding modes or cooperative binding on long DNA substrates. These features are unique to Pf-SSB, making it an ideal tool to probe the presence of free ssDNA in any biochemical reaction. Using this Pf-SSBMDCC probe as a sensor for free ssDNA, we have investigated the clearing of preformed yeast Rad51 nucleoprotein filaments by the Srs2 helicase during HR. Our studies provide a rate for the disassembly of the Rad51 filament by full length Srs2 on long ssDNA substrates. Mutations in the conserved 2B domain in the homologous bacterial UvrD, Rep and PcrA helicases show an enhancement of DNA unwinding activity, but similar mutations in Srs2 do not affect its DNA unwinding or Rad51 clearing properties. These studies showcase the utility of the Pf-SSB probe in mechanistic investigation of enzymes that function in DNA metabolism.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Davenport, Eric Parker; Harris, Derek F.; Origanti, Sofia; and Antony, Edwin, "Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent Plasmodium falciparum SSB as a Reporter for Single-Stranded DNA" (2016). Biological Sciences Faculty Research and Publications. 526.
https://epublications.marquette.edu/bio_fac/526
Comments
Published version. PLoS One, Vol. 11, No. 7 (July 14, 2016). DOI. © 2016 Davenport et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.