Localization of RecA in Escherichia coli K-12 using RecA-GFP.
RecA is important in recombination, DNA repair and repair of replication forks. It functions through the production of a protein-DNA filament. To study the localization of RecA in live Escherichia coli cells, the RecA protein was fused to the green fluorescence protein (GFP). Strains with this gene have recombination/DNA repair activities three- to tenfold below wild type (or about 1000-fold above that of a recA null mutant). RecA-GFP cells have a background of green fluorescence punctuated with up to five foci per cell. Two types of foci have been defined: 4,6-diamidino-2-phenylindole (DAPI)-sensitive foci that are bound to DNA and DAPI-insensitive foci that are DNA-less aggregates/storage structures. In log phase cells, foci were not localized to any particular region. After UV irradiation, the number of foci increased and they localized to the cell centre. This suggested colocalization with the DNA replication factory. recA, recB and recF strains showed phenotypes and distributions of foci consistent with the predicted effects of these mutations.
Renzette
Nicholas
N
Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, MA 01003, USA.
Gumlaw
Nathan
N
Nordman
Jared T
JT
Krieger
Marlee
M
Yeh
Su-Ping
SP
Long
Edward
E
Centore
Richard
R
Boonsombat
Ruethairat
R
Sandler
Steven J
SJ
D002876:Chromosomes, Bacterial / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism / Q000528:radiation effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D009154:Mutation; D011938:Rec A Recombinases / Q000032:analysis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D011995:Recombination, Genetic; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011938:Rec A Recombinases