TY - JOUR
T1 - A new trick for an old dog
T2 - Tray binding to a homopurine-homopyrimidine run attenuates DNA replication
AU - Krasilnikova, Maria M.
AU - Smirnova, Ekaterina V.
AU - Krasilnikov, Andrei S.
AU - Mirkin, Sergei M.
N1 - Funding Information:
We are indebted to Elena Siyanova, who was the first to suggest that our replication data could be explained by protein binding rather than triplex formation. We thank Elena Davydova for her invaluable help in TraY purification, George Samadashwily for his input in the early stage of these experiments, Pradip Raychadhury, Pavel Shiyanov and Tatiana Shiyanova for their help with chromatography, Carol Beach for expert protein sequencing, Igor Panyutin for helpful discussions and Gerald Buldak for proofreading the manuscript. This work was supported by grant GM60987 from the NIH to S.M.M. E.V.S. is supported by a graduate fellowship from the University of Illinois at Chicago.
PY - 2001/10/19
Y1 - 2001/10/19
N2 - 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.
AB - 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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U2 - 10.1006/jmbi.2001.5059
DO - 10.1006/jmbi.2001.5059
M3 - Article
C2 - 11800556
AN - SCOPUS:0035914472
SN - 0022-2836
VL - 313
SP - 271
EP - 282
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -