A new trick for an old dog: TraY binding to a homopurine-homopyrimidine run attenuates DNA replication.
The effects of the d(GA)(n).d(TC)(n) repeat on plasmid replication in Escherichia coli cells were analyzed using electrophoretic analysis of replication intermediates. This repeat appeared to stall the replication fork progression in E. coli strains carrying F' episomes. The potency of replication stalling increased with the repeat's length but did not depend on its orientation relative to the replication origin, or transcription through the repeat. Treatment of E. coli cells with the protein synthesis inhibitor chloramphenicol abolished replication blockage, indicating that protein binding might be responsible for the repeat-caused replication blockage. Concordantly, dimethylsulfate footprinting in vivo revealed methylation protection of all guanine residues within the d(GA)(n).d(TC)(n). Gel retardation assays with crude cell extracts confirmed the presence of a d(GA)(n).d(TC)(n) -binding activity in F', but not F(-), strains. Further, strains cured from the F' episome lost this activity, while F(-) strains that acquired the F' factor via conjugation, acquired d(GA)(n).d(TC)(n)-binding activity as well. Thus, this d(GA)(n).d(TC)(n)-binding protein is encoded by the F' factor. Purification of this protein by affinity chromatography revealed a single polypeptide with an apparent molecular mass of 15.2 kDa. Microsequencing of its two tryptic peptides revealed two perfect matches with the TraY protein, which is encoded by the F factor. Overexpression of an individual TraY protein in the F(-) E. coli strain conveyed d(GA)(n).d(TC)(n)-binding activity in vitro and replication stalling at d(GA)(n).d(TC)(n) repeats in vivo. We conclude that TraY binding to a homopurine-homopyrimidine repeat is responsible for stalling DNA replication. Biological applications of this phenomenon are discussed.
Krasilnikova
M M
MM
Department of Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.
Smirnova
E V
EV
Krasilnikov
A S
AS
Mirkin
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002701:Chloramphenicol / Q000494:pharmacology; D002846:Chromatography, Affinity; D003227:Conjugation, Genetic / Q000235:genetics; D018983:DNA Footprinting; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004285:Dogs; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013463:Sulfuric Acid Esters / Q000378:metabolism
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D013463:Sulfuric Acid Esters; C063238:traY protein, E coli; D002701:Chloramphenicol; C007482:dimethyl sulfate