Design and evaluation of a tryptophanless RecA protein with wild type activity.
The C-terminal domain of the Escherichia coli RecA protein contains two tryptophan residues whose native fluorescence emission provides an interfering background signal when other fluorophores such as 1,N(6)-ethenoadenine, 2-aminopurine and other tryptophan residues are used to probe the protein's activities. Replacement of the wild type tryptophans with nonfluorescent residues is not trivial because one tryptophan is highly conserved and the C-terminal domain functions in both DNA binding as well as interfilament protein-protein contact. We undertook the task of creating a tryptophanless RecA protein with WT RecA activity by selecting suitable amino acid replacements for Trp290 and Trp308. Mutant proteins were screened in vivo using assays of SOS induction and cell survival following UV irradiation. Based on its activity in these assays, the W290H-W308F W-less RecA was purified for in vitro characterization and functioned like WT RecA in DNA-dependent ATPase and DNA strand exchange assays. Spectrofluorometry indicates that the W290H-W308F RecA protein generates no significant emission when excited with 295-nm light. Based on its ability to function as wild type protein in vivo and in vitro, this dark RecA protein will be useful for future fluorescence experiments.
Berger
M D
MD
Department of Chemistry, Rice University, Houston, Texas 77005, USA.
Lee
A M
AM
Simonette
R A
RA
Jackson
B E
BE
Roca
A I
AI
Singleton
S F
SF
D015075:2-Aminopurine / Q000494:pharmacology; D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002470:Cell Survival; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009153:Mutagens; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D009153:Mutagens; C004981:1,N(6)-ethenoadenine; D015075:2-Aminopurine; D014364:Tryptophan; D004247:DNA; D011938:Rec A Recombinases; D000225:Adenine