Faculty Collaboration Database

Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent Plasmodium falciparum SSB as a Reporter for Single-Stranded DNA. PLoS One 2016;11(7):e0159242

Date

07/16/2016

Pubmed ID

27416037

Pubmed Central ID

PMC4945038

DOI

10.1371/journal.pone.0159242

Scopus ID

2-s2.0-84978512149 (requires institutional sign-in at Scopus site)   3 Citations

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.

Author List

Davenport EP, Harris DF, Origanti S, Antony E

Author

Edwin Antony PhD Assistant Professor in the Biology department at Marquette University

MESH terms used to index this publication - Major topics in bold

Coumarins
DNA Probes
DNA, Single-Stranded
DNA-Binding Proteins
Fluorescence
Nucleoproteins
Plasmodium falciparum
Rad51 Recombinase
Saccharomyces cerevisiae Proteins