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 Esc herichia coli cells were analyzed using electrophoretic analysis of replica tion intermediates. This repeat appeared to stall the replication fork prog ression in E. coli strains carrying F' episomes. The potency of replication stalling increased with the repeat's length but did not depend on its orie ntation relative to the replication origin, or transcription through the re peat. Treatment of E. coli cells with the protein synthesis inhibitor chlor amphenicol abolished replication blockage, indicating that protein binding might be responsible for the repeat-caused replication blockage. Concordant ly, dimethylsulfate footprinting in vivo revealed methylation protection of all guanine residues within the d(GA)(n)(.)d(TC)(n). Gel retardation assay s 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 t he F' episome lost this activity, while F- strains that acquired the F' fac tor 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 o f its two tryptic peptides revealed two perfect matches with the TraY prote in, which is encoded by the F factor. Overexpression of an individual TraY protein in the F (-) E. coli strain conveyed d(GA), - d(TC),,-binding activ ity in vitro and replication stalling at d(GA)(n)(.)d(TC)(n) repeats in viv o. We conclude that TraY binding to a homopurine-homopyrimidine repeat is r esponsible for stalling DNA replication. Biological applications of this ph enomenon are discussed. (C) 2001 Academic Press.