In vitro Studies of DNA Replication in Bacillus subtilis and Phage SPP1

Zinder, John Charles

Graduate Thesis Or Dissertation

In vitro Studies of DNA Replication in Bacillus subtilis and Phage SPP1 Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/rf55z8078

Abstract

Chromosomal DNA replication is an essential and demanding task that all cells must undertake in order to proceed with division. Decades of research have made Escherichia coli (Eco) the prototype for the study of DNA replication and many of the lessons learned from the study of the Eco replisome, the term for the proteins that carries out replication, have held true in other organisms. DNA replication in Bacillus subtilis (Bsu), a bacterium that diverged from Eco approximately 1.5 billion years ago, follows many of the same general principles of Eco with the important difference being that it uses two DNA polymerases rather than one. One polymerase, PolC, performs leading strand synthesis as well as the majority of lagging strand synthesis while the other polymerase, DnaE, is responsible for only a short extension of an RNA primer at the lagging strand before handing off synthesis to PolC to finish the Okazaki fragment. The details of this handoff have been examined using in vitro primer extension assays. It was found that DnaE is sufficiently processive that it must be actively evicted from the primer terminus in order for the exchange to occur. Additionally, in vitro studies of the replisome of phage SPP1, a virulent dsDNA phage that infects Bsu, have been performed. It was found that a phage encoded ssDNA binding protein was able to inhibit the PolC polymerase in the context of the host replisome but not the phage replisome, providing a novel mechanism through which a phage can subvert the host replication machinery while ensuring use of its own. These studies expand on the already rich repertoire of known strategies for cellular and viral DNA replication, and lay the way for more detailed studies of their precise mechanism.

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