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J Biol Chem, Vol. 275, Issue 18, 13529-13534, May 5, 2000
From the Department of Molecular Genetics and Biochemistry,
University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261
Plasmids of the pT181 family encode initiator
proteins that act as dimers during plasmid rolling circle (RC)
replication. These initiator proteins bind to the origin of replication
through a sequence-specific interaction and generate a nick at the
origin that acts as the primer for RC replication. Previous studies
have demonstrated that the initiator proteins contain separate DNA binding and nicking-closing domains, both of which are required for
plasmid replication. The tyrosine residue at position 191 of the
initiator RepC protein of pT181 is known to be involved in nicking at
the origin. We have generated heterodimers of RepC that consist of
different combinations of wild type, DNA binding, and nicking mutant
monomers to identify the role of each of the two monomers in RC
replication. One monomer with DNA binding activity was sufficient for
the targeting of the initiator to the origin, and the presence of
Tyr-191 in one monomer was sufficient for the initiation of
replication. On the other hand, a dimer consisting of one monomer
defective in DNA binding and the other defective in origin nicking
failed to initiate replication. Our results demonstrate that the
monomer that promotes sequence-specific binding to the origin must also
nick the DNA to initiate replication. Interestingly, whereas Tyr-191 of
the initiator was required for nicking at the origin to initiate
replication, it was dispensable for termination, suggesting that
alternate amino acids in the initiator may promote termination but not initiation.
Role of Individual Monomers of a Dimeric Initiator Protein in the
Initiation and Termination of Plasmid Rolling Circle Replication*
*
This work was supported by National Institutes of Health
Grant GM31685 (to S. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Genetics and Biochemistry, University of Pittsburgh School of Medicine,
Rm. East 1240 Biomedical Science Tower, Pittsburgh, PA 15261. Tel.:
412-648-9025; Fax: 412-624-1401; E-mail: Khan@pitt.edu.
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