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RESEARCH

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Recognition of the replication origin (ori) by initiator protein is a recurring theme for the regulated initiation of DNA replication in diverse biological systems. The objective of the work carried out in our laboratory is to understand the initiation process and its regulation.

Our model system is the gamma-ori of the antibiotic-resistance plasmid R6K. Control of gamma-ori initiation is determined both by plasmid-encoded and host-encoded factors. The two central regulatory elements of the plasmid are a multifunctional initiator protein pi, and sequence-related DNA target sites, the inverted half-repeats (IRs) and the direct repeats (DRs), collectively called iterons. The replication activator and inhibitor activities of pi seem to be at least partially distributed between two naturally occurring pi polypeptides (designated by their molecular weights pi35.0 and pi30.5).

Regulatory variants of pi with altered states of oligomerization in nucleoprotein complexes with DRs have been isolated in our laboratory. The properties of these mutants laid the foundation for our model of pi protein activity which proposes that different protein surfaces are required for the formation of functionally distinct complexes of pi with DRs and IRs. These mutants also suggest that pi variants have a modular structure; the C-terminus contains the DNA-binding domain while the N-terminus controls protein oligomerization. Additionally, pi35.0 binds to a non-iteron DNA sequence in the A+T-rich segment of gamma-ori. That binding site is at or near the site from which synthesis of the leading strand begins.

Our current model proposes that the regulation of gamma-ori replication is accomplished through the ability of the multifunctional pi protein to act as an initiator and an inhibitor of replication as well as a repressor of its own synthesis.

The following mechanisms have been demonstrated or implicated to play a role in the regulation of this system:

* Dimers of the pi35.0 protein bind to the pir gene operator IR auto-repressing synthesis of pi35.0 and pi30.5.

* Monomers of the pi35.0 bind to DR’s to open the DNA helix and activate gamma-ori replication.

* Dimers pi35.0 and pi30.5 compete with monomers of pi35.0 and thereby inhibit activation of gamma-ori.

* The binding of pi35.0 dimers the A+T-rich region may prevent recruitment and or activity of proteins necessary for priming.

In addition to the requirement of pi protein, gamma-ori replication depends on many cellular proteins which bind directly to the gamma-ori DNA (see figure). Continuing investigations focus on a detailed examination of the function of the E. coli host proteins DnaA, IHF and Fis in the control of gamma-ori replication.

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Understanding the replication control of bacterial plasmids is an endeavor of fundamental importance both in terms of basic biological science and practical application. The societal usefulness of our work arises from the fact that plasmids are at the center of a variety of important medical, agricultural and biotechnological phenomena. Plasmid R6K carries genes for penicillin and streptomycin resistance. Thus, the regulation of R6K replication by pi protein plays a role in the spread of antibiotic resistance. An alarming rise in the incidence of antibiotic resistance is rapidly undermining the current treatment methods for many pathogens. A thorough understanding of the structure/function relationship(s) of plasmids conferring antibiotic resistence may ultimately allow us to prevent their spread in the environment.

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