@article {809,
	title = {Single molecule analysis of a red fluorescent RecA protein reveals a defect in nucleoprotein filament nucleation that relates to its reduced biological functions.},
	journal = {J Biol Chem},
	volume = {284},
	year = {2009},
	month = {2009 Jul 10},
	pages = {18664-73},
	abstract = {Fluorescent fusion proteins are exceedingly useful for monitoring protein localization in situ or visualizing protein behavior at the single molecule level. Unfortunately, some proteins are rendered inactive by the fusion. To circumvent this problem, we fused a hyperactive RecA protein (RecA803 protein) to monomeric red fluorescent protein (mRFP1) to produce a functional protein (RecA-RFP) that is suitable for in vivo and in vitro analysis. In vivo, the RecA-RFP partially restores UV resistance, conjugational recombination, and SOS induction to recA(-) cells. In vitro, the purified RecA-RFP protein forms a nucleoprotein filament whose k(cat) for single-stranded DNA-dependent ATPase activity is reduced approximately 3-fold relative to wild-type protein, and which is largely inhibited by single-stranded DNA-binding protein. However, RecA protein is also a dATPase; dATP supports RecA-RFP nucleoprotein filament formation in the presence of single-stranded DNA-binding protein. Furthermore, as for the wild-type protein, the activities of RecA-RFP are further enhanced by shifting the pH to 6.2. As a consequence, RecA-RFP is proficient for DNA strand exchange with dATP or at lower pH. Finally, using single molecule visualization, RecA-RFP was seen to assemble into a continuous filament on duplex DNA, and to extend the DNA approximately 1.7-fold. Consistent with its attenuated activities, RecA-RFP nucleates onto double-stranded DNA approximately 3-fold more slowly than the wild-type protein, but still requires approximately 3 monomers to form the rate-limited nucleus needed for filament assembly. Thus, RecA-RFP reveals that its attenuated biological functions correlate with a reduced frequency of nucleoprotein filament nucleation at the single molecule level.},
	keywords = {Cell Nucleus, DNA, DNA, Single-Stranded, Escherichia coli, Hydrogen-Ion Concentration, Kinetics, Luminescent Proteins, Nucleoproteins, Plasmids, Protein Binding, Rec A Recombinases, Recombination, Genetic, Sensitivity and Specificity, Ultraviolet Rays},
	issn = {0021-9258},
	doi = {10.1074/jbc.M109.004895},
	author = {Handa, Naofumi and Amitani, Ichiro and Gumlaw, Nathan and Sandler, Steven J and Kowalczykowski, Stephen C}
}
@article {824,
	title = {A dnaT mutant with phenotypes similar to those of a priA2::kan mutant in Escherichia coli K-12.},
	journal = {Genetics},
	volume = {167},
	year = {2004},
	month = {2004 Jun},
	pages = {569-78},
	abstract = {The ability to repair damaged replication forks and restart them is important for cell survival. DnaT is essential for replication restart in vitro and yet no definite genetic analysis has been done in Escherichia coli K-12. To begin, dnaT822, an in-frame six-codon (87-92) deletion was constructed. DnaT822 mutants show colony size, cell morphology, inability to properly partition nucleoids, UV sensitivity, and basal SOS expression similar to priA2::kan mutants. DnaT822 priA2::kan double mutants had phenotypes similar to those of the single mutants. DnaT822 and dnaT822 priA2::kan mutant phenotypes were fully suppressed by dnaC809. Previously, a dominant temperature-sensitive lethal mutation, dnaT1, had been isolated in E. coli 15T(-). DnaT1 was found to have a base-pair change relative to the E. coli 15T(-) and E. coli K-12 dnaT genes that led to a single amino acid change: R152C. A plasmid-encoded E. coli K-12 mutant dnaT gene with the R152C amino acid substitution did not display a dominant temperature-sensitive lethal phenotype in a dnaT(+) strain of E. coli K-12. Instead, this mutant dnaT gene was found to complement the E. coli K-12 dnaT822 mutant phenotypes. The significance of these results is discussed in terms of models for replication restart.},
	keywords = {Base Sequence, Codon, DNA Primers, DNA Repair, DNA Replication, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli K12, Escherichia coli Proteins, Molecular Sequence Data, Mutation, Plasmids, Polymerase Chain Reaction, Sequence Deletion},
	issn = {0016-6731},
	doi = {10.1534/genetics.103.025296},
	author = {McCool, Jesse D and Ford, Christopher C and Sandler, Steven J}
}
