TY - JOUR
T1 - Multiple DNA conformational changes induced by an initiator protein precede the nicking reaction in a rolling circle replication origin
AU - Higashitani, Atsushi
AU - Greenstein, David
AU - Hirokawa, Hideo
AU - Asano, Satoshi
AU - Horiuchi, Kensuke
PY - 1994/4/7
Y1 - 1994/4/7
N2 - The core origin for plus strand DNA replication of filamentous bacteriophage f1 binds the initiator protein (gpII), which subsequently introduces a specific nick in the plus strand. The core origin consists of a nicking region and a binding region. The binding of gpII occurs in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II). Results of gel retardation experiments using circularly permuted DNA fragments and direct visualization by electron microscopy show that gpII induces successive bends within the binding region upon formation of the complexes. We show that gpII binding induces duplex melting in the nicking region using KMnO4 modification of unpaired thymidine residues as a probe for melting. Origin binding occurred in the absence of superhelicity of DNA and Mg2+, whereas duplex melting required superhelical DNA, but not Mg2+. Deletion analyses indicated that hypothetical formation of a cruciform around the nicking site is not necessary for either melting or nicking. A mutation in gpII resulted in stimulation of duplex melting and nicking without showing obvious effects on bending. This suggests that the mechanism of melting involves local interaction between gpII and the nicking region. Furthermore, using synthetic oligonucleotide substrates, we show that the nicking reaction takes place efficiently when the nicking region is single-stranded and the binding region is double-stranded. These results indicate that the nicking reaction is preceded by an ordered series of protein-induced DNA-conformational changes: successive bending of the origin upon gpII binding, followed by duplex melting that requires negative superhelicity.
AB - The core origin for plus strand DNA replication of filamentous bacteriophage f1 binds the initiator protein (gpII), which subsequently introduces a specific nick in the plus strand. The core origin consists of a nicking region and a binding region. The binding of gpII occurs in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II). Results of gel retardation experiments using circularly permuted DNA fragments and direct visualization by electron microscopy show that gpII induces successive bends within the binding region upon formation of the complexes. We show that gpII binding induces duplex melting in the nicking region using KMnO4 modification of unpaired thymidine residues as a probe for melting. Origin binding occurred in the absence of superhelicity of DNA and Mg2+, whereas duplex melting required superhelical DNA, but not Mg2+. Deletion analyses indicated that hypothetical formation of a cruciform around the nicking site is not necessary for either melting or nicking. A mutation in gpII resulted in stimulation of duplex melting and nicking without showing obvious effects on bending. This suggests that the mechanism of melting involves local interaction between gpII and the nicking region. Furthermore, using synthetic oligonucleotide substrates, we show that the nicking reaction takes place efficiently when the nicking region is single-stranded and the binding region is double-stranded. These results indicate that the nicking reaction is preceded by an ordered series of protein-induced DNA-conformational changes: successive bending of the origin upon gpII binding, followed by duplex melting that requires negative superhelicity.
KW - DNA bending
KW - DNA replication
KW - Duplex melting
KW - Filamentous bacteriophage
KW - Negative superhelicity
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U2 - 10.1006/jmbi.1994.1242
DO - 10.1006/jmbi.1994.1242
M3 - Article
C2 - 8151700
AN - SCOPUS:0028364238
SN - 0022-2836
VL - 237
SP - 388
EP - 400
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -