Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase.
The protozoan parasite responsible for human amoebiasis is Entamoeba histolytica. An important facet of the life cycle of E. histolytica involves the conversion of the mature trophozoite to a cyst. This transition is thought to involve homologous recombination (HR), which is dependent upon the Rad51 recombinase. Here, a biochemical characterization of highly purified ehRad51 protein is presented. The ehRad51 protein preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. Although the homologous DNA pairing activity of ehRad51 is weak, it is strongly enhanced by the presence of two HR accessory cofactors, calcium and Hop2-Mnd1. The biochemical system described herein was used to demonstrate the potential for targeting ehRad51 with two small molecule inhibitors of human RAD51. We show that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited ehRad51 by interfering with DNA binding and attenuated encystation in Entamoeba invadens, while B02 had no effect on ehRad51 strand exchange activity. These results provide insight into the underlying mechanism of homology-directed DNA repair in E. histolytica.
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA; Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, SC 29634, USA.
Andrew A
AA
Kelso
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA; Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, SC 29634, USA.
Steven D
SD
Goodson
Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, SC 29634, USA; Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA.
Suchitra
S
Chavan
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
Amanda F
AF
Say
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
Audrey
A
Turchick
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
Deepti
D
Sharma
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
LeAnna L
LL
Ledford
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
Erin
E
Ratterman
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
Kristin
K
Leskoske
Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA.
Ada V
AV
King
Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA.
Christopher C
CC
Attaway
Center for Optical Materials Science and Engineering Technologies, Clemson University, Clemson, SC 29634, USA; Department of Material Science and Engineering, Clemson University, Clemson, SC 29634, USA.
Yura
Y
Bandera
Center for Optical Materials Science and Engineering Technologies, Clemson University, Clemson, SC 29634, USA; Department of Material Science and Engineering, Clemson University, Clemson, SC 29634, USA.
Stephen H
SH
Foulger
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA.
Alexander V
AV
Mazin
Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, SC 29634, USA; Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA; Clemson University School of Health Research, Clemson, SC 29634, USA.
Lesly A
LA
Temesvari
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA; Center for Optical Materials Science and Engineering Technologies, Clemson University, Clemson, SC 29634, USA; Clemson University School of Health Research, Clemson, SC 29634, USA. Electronic address: msehorn@clemson.edu.
Michael G
MG
Sehorn
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004748:Entamoeba histolytica / Q000201:enzymology*; D004789:Enzyme Activation; D059765:Homologous Recombination; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins; D013379:Substrate Specificity
D002352:Carrier Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D051135:Rad51 Recombinase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium
DNA repair; Entamoeba histolytica; Homologous recombination; RAD51; Recombinase inhibitors
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