A new trick for an old dog

Tray binding to a homopurine-homopyrimidine run attenuates DNA replication

Maria Krasilnikova, Ekaterina V. Smirnova, Andrey S. Krasilnikov, Sergei M. Mirkin

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The effects of the d(GA)n·d(TC)nrepeat 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)nGel retardation assays with crude cell extracts confirmed the presence of a d(GA)n·d(TC)n-binding activity in F′, but not Fstrains. Further, strains cured from the F′ episome lost this activity, while Fstrains 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 15.2kDa. 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 FE.coli strain conveyed d(GA)n·d(TC)n-binding activity in vitro and replication stalling at d(GA)n·d(TC)nrepeats 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.

Original languageEnglish (US)
Pages (from-to)271-282
Number of pages12
JournalJournal of Molecular Biology
Volume313
Issue number2
DOIs
StatePublished - Oct 19 2001

Fingerprint

F Factor
DNA Replication
Plasmids
Dogs
Escherichia coli
Biological Phenomena
Peptides
Proteins
Protein Synthesis Inhibitors
Replication Origin
Guanine
Chloramphenicol
Cell Extracts
Complex Mixtures
Affinity Chromatography
Protein Binding
Methylation
Carrier Proteins

All Science Journal Classification (ASJC) codes

  • Molecular Biology

Cite this

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title = "A new trick for an old dog: Tray binding to a homopurine-homopyrimidine run attenuates DNA replication",
abstract = "The effects of the d(GA)n·d(TC)nrepeat 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)nGel retardation assays with crude cell extracts confirmed the presence of a d(GA)n·d(TC)n-binding activity in F′, but not Fstrains. Further, strains cured from the F′ episome lost this activity, while Fstrains 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 15.2kDa. 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 FE.coli strain conveyed d(GA)n·d(TC)n-binding activity in vitro and replication stalling at d(GA)n·d(TC)nrepeats 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.",
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A new trick for an old dog : Tray binding to a homopurine-homopyrimidine run attenuates DNA replication. / Krasilnikova, Maria; Smirnova, Ekaterina V.; Krasilnikov, Andrey S.; Mirkin, Sergei M.

In: Journal of Molecular Biology, Vol. 313, No. 2, 19.10.2001, p. 271-282.

Research output: Contribution to journalArticle

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