Binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid R6K

The Journal of Biological Chemistry
Selvi KunnimalaiyaanMarcin Filutowicz

Abstract

Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the gamma origin of plasmid R6K, the Rep protein, pi, is distinctive in that it can bind the seven 22-bp iterons in two forms; pi monomers activate replication, whereas pi dimers act as inhibitors. In this work, we used wild type and variants of the pi protein with altered monomer/dimer ratios to study iteron/pi interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by pi monomers and dimers. We also isolated iteron mutants that affected the binding of pi monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, pi monomers interact with nucleotides spanning the entire length of the iteron. In contrast, pi dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomer...Continue Reading

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Citations

Mar 12, 2013·Plasmid·Sheryl A Rakowski, Marcin Filutowicz
May 20, 2005·Journal of Bacteriology·Selvi KunnimalaiyaanMarcin Filutowicz
Mar 27, 2007·Journal of Molecular Biology·Lisa M BowersMarcin Filutowicz
Nov 19, 2011·FEMS Microbiology Reviews·Magdalena RajewskaIgor Konieczny
Feb 26, 2008·Journal of Molecular Biology·Lisa M Bowers, Marcin Filutowicz
Dec 22, 2006·Biochemical and Biophysical Research Communications·Man HanTateo Itoh
May 4, 2021·Microbial Biotechnology·Rebecca L DineenDouwe van Sinderen

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