Isolation and characterization of mutants of Tus, the replication arrest protein of Escherichia coli.
Mutations in the tus gene of Escherichia coli, which encodes the replication arrest protein Tus, were isolated using a selection scheme based on the plasmid pHV750T2+, which transforms tus mutants at a much higher frequency than wild type strains. Seven mutants containing single nucleotide substitutions were isolated, and all of these mutants showed reduced or complete loss of DNA binding and replication arrest activity. Two of the mutant proteins, containing a valine (A173V) or threonine (A173T) in place of the alanine normally found at amino acid 173, were purified and characterized further. A173T had a 4100-fold lower affinity for Ter sites than wild type Tus and was unable to halt DNA replication in vivo or inhibit DnaB-catalyzed strand displacement in an in vitro helicase assay. A173V showed a 130-fold lower affinity for Ter sites than wild type Tus but was still able to arrest DNA replication in vivo, suggesting that protein-protein interactions were responsible for Tus-mediated arrest of DNA replication. In addition, we found that A173V was a weak inhibitor of DnaB-catalyzed strand displacement in vitro, yet halted DNA replication in vivo at 75% of the efficiency of wild type Tus. We concluded from these observations that the standard in vitro helicase assay was inadequate for measuring Tus activity.
Skokotas
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A
Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104.
Wrobleski
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Hill
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TM
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D002918:Chymotrypsin; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C061910:tus protein, Bacteria; C489236:tus protein, E coli; D002918:Chymotrypsin; D004265:DNA Helicases