Open AccessUncoupling of the DNA topoisomerase and replication activities of an initiator protein.
Author(s) -
Laurie A Dempsey,
Patrick Birch,
Saleem A. Khan
Publication year - 1992
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.89.7.3083
Subject(s) - topoisomerase , dna replication , biology , origin recognition complex , seqa protein domain , dna , eukaryotic dna replication , replication factor c , microbiology and biotechnology , primer (cosmetics) , mutagenesis , biochemistry , replication protein a , rolling circle replication , control of chromosome duplication , dna binding protein , gene , mutant , chemistry , transcription factor , organic chemistry
The replication initiator proteins encoded by the pT181 and related plasmids have sequence-specific DNA binding and topoisomerase activities. These proteins create a site-specific nick in one strand of the DNA at the origin of replication that serves as a primer for the initiation of replication. To define the regions of the pT181-encoded initiator protein, RepC, that are involved in its DNA binding, topoisomerase, and replication activities, we have carried out site-directed mutagenesis of the repC gene. Analysis of mutant RepC proteins in vitro and in vivo has identified the amino acids that are critical for its various biochemical activities. The DNA binding domain of RepC was found to be located near its C-terminal region and was different from the domain involved in its sequence-specific topoisomerase activity. These studies also showed that the DNA topoisomerase activity of the initiator protein can be uncoupled from its tight noncovalent DNA binding and replication activities.