Site-directed mutagenesis and phylogenetic comparisons of the Escherichia coli Tus protein: DNA-protein interactions alone can not account for Tus activity.
The Tus protein of Escherichia coli is capable of arresting DNA replication in an orientation-dependent manner when bound to specific sequences in the bacterial chromosome called Ter sites. Arrest of DNA replication has been postulated to occur either by a barrier mechanism, where Tus acts as a physical block to replication fork progression, or through protein-protein interactions between Tus and some component of the replication fork. A previous mutational analysis of Tus suggested that the amino acids in the L1 loop might play a role in replication arrest. Site-directed mutagenesis of amino acids in the L1 loop and other amino acid residues on the "non-permissive" face of Tus was performed to identify residues that affected Tus function. One mutant, E47Q, gave results that are inconsistent with the barrier model, showing a greater affinity for the Ter site (with a t 1/2 of 348 min versus 150 min for wild-type Tus) but a reduced ability to arrest DNA replication in vivo. In addition to the site-directed mutagenesis studies, the tus genes of Salmonella, Klebsiella, and Yersinia were sequenced and the proteins expressed in E. coli to assess their ability to arrest DNA replication. The results presented here support a role for protein-protein interactions in Tus function, and suggest that residues E47 and E49 participate in replication fork arrest.
Henderson
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Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks 58202-9037, USA.
Nilles
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Valjavec-Gratian
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Hill
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TM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004755:Enterobacteriaceae / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D007709:Klebsiella / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011513:Proteus mirabilis / Q000235:genetics; D012477:Salmonella enteritidis / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000235:genetics; D015008:Yersinia enterocolitica / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061910:tus protein, Bacteria; C489236:tus protein, E coli