In the circular Escherichia coli chromosome, two replisomes are assembled at the unique origin of replication and drive DNA synthesis in opposite directions until they meet in the terminus region across from the origin. Despite the difference in rates of the two replisomes, their arrival at the terminus is synchronized through a highly specialized system consisting of the terminator protein (Tus) bound to the termination sites (Ter). This synchronicity is mediated by the polarity of the Tus−Ter complex that stops replisomes from one direction (non-permissive face) but not the other (permissive face). Two oppositely oriented clusters of five Tus–Ters that each block one of the two replisomes create a “replication fork trap” for the first arriving replisome while waiting for the late arriving one. Despite extensive biochemical and structural studies, the molecular mechanism behind Tus−Ter polar arrest activity remained controversial. Moreover, none of the previous work provided answers for the long-standing discrepancy between the ability of Tus−Ter to permanently stop replisomes in vitro and its low efficiency in vivo. Here, I spearheaded a collaborative project that combined single-molecule DNA replication assays, X-ray crystallography and binding studies to provide a true molecular-level understanding of the underlying mechanism of Tus−Ter polar arrest activity. We showed that efficiency of Tus−Ter is determined by a head-to-head kinetic competition between rate of strand separation by the replisome and rate of rearrangement of Tus−Ter interactions during the melting of the first 6 base pairs of Ter. This rearrangement maintains Tus’s strong grip on the DNA and stops the advancing replisome from breaking into Tus−Ter central interactions, but only transiently. We further showed how this kinetic competition functions within the context of two mechanisms to impose permanent fork stoppage. The rate-dependent fork arrest activity of Tus−Ter explains its low efficiency in vivo and why contradictory in vitro results from previous studies have led to controversial elucidations of the mechanism. It also provides the first example where the intrinsic heterogeneity in rate of individual replisomes could have different biological outcomes in its communication with double-stranded DNA-binding protein barriers.

A DNA fragment containing the coding region for gene 6 of Bacterial phage ϕ29 was placed into an expression vector. The ϕ29 gene 6 protein was isolated in large amounts by chromatography on double-stranded DNA cellulose and DE52 cellulose. The ϕ29 gene 6 protein was determined to be greater 95% pure and has a molecular weight of approximately 16,000. The ϕ29 gene 6 protein is thought to be a dimer in its native form. The partial N-terminal amino acid sequence of the purified protein is identically to the inferred amino acid sequence from the nucleotide sequence of ϕ29 gene 6. Gene 6 protein of ϕ29 aggregates in a more purified state which suggest protein to protein interactions. Purified gene 6 protein did not stimulate the ϕ29 in vitro DNA replication system and may require binding with other replication proteins to enable it to function. Gene 6 protein binds weakly to double-stranded and single-strand DNA cellulose. There is segmental amino acid sequence and secondary structure homology with adenovirus DNA binding protein Antibody to gene 6 protein inhibits it from binding to ϕ29 DNA. The results presented in this dissertation suggest that ϕ29 gene 6 protein is a weak DNA bind protein and may not be required for the in vitro ϕ29 DNA replication system.

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