Mutations in the Escherichia coli Tus protein define a domain positioned close to the DNA in the Tus-Ter complex.
A new genetic screen for mutations in the tus gene of Escherichia coli has been devised that selects for Tus proteins with altered ability to arrest DNA replication. We report here the characterization of three such mutants: TusP42S, TusE49K, and TusH50Y. TusP42S and TusE49K arrest DNA replication in vivo at 36% of the efficiency of wild-type Tus, whereas TusH50Y functions at 78% efficiency. The loss of replication arrest activity did not correlate with changes in the stability of the Tus-TerB complexes formed by the mutant proteins. TusE49K formed a more stable protein-DNA complex than wild-type Tus (t1/2 of 178 versus 149 min, respectively) and TusP42 had a 9-min half-life, yet these two mutants showed identical efficiencies for replication arrest. When tested in vitro using a helicase assay or an oriC replication system, we observed a general, but imperfect, correlation between the in vivo and in vitro assays. Finally, the half-lives of the mutant protein-DNA complexes suggested that the domain of Tus where these mutations are located is positioned close to the DNA in the Tus-Ter complex.
Skokotas
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A
Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104, USA.
Hiasa
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H
Marians
K J
KJ
O'Donnell
L
L
Hill
T M
TM
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004261:DNA Replication / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D018741:Replication Origin
D001426:Bacterial Proteins; C062011:DNA replication terminus site-binding protein, E coli; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061910:tus protein, Bacteria; C489236:tus protein, E coli