Degree Name

Doctor of Philosophy


School of Chemistry


This thesis presents analyses of key protein-protein and protein-nucleic acid interactions in the E. coli replication machinery. The E. coli single-stranded DNA-binding protein (SSB) is a central mediator of DNA metabolism, binding to regions of exposed single-stranded DNA (ssDNA) and playing crucial roles in DNA replication, recombination and repair. SSB is a stable homotetramer, held together by interactions between the N-terminal domains of its subunits. Structurally, the N-terminal domains form classic oligonucleotide-binding folds (OB-folds), and are also responsible for ssDNA binding. The SSB-ssDNA complexes may adopt a number of configurations, differing in the extent of SSB-ssDNA contact, and are heavily influenced by salt concentration and protein- DNA ratio. The C-terminus of SSB appears to be unstructured, and its highly conserved tip (~eight residues) is responsible for interacting with a host of other proteins involved in DNA metabolism and regulating their functions. Recent evidence suggests that the SSB C-termini, in the absence of bound DNA, may be able to occupy the OB-folds of the tetramer via an electrostatic interaction.

The first aim of this thesis was to use electrospray ionisation mass spectrometry (ESI-MS) to characterise SSB and its complexes with ssDNA, and to assess the validity of ESI-MS for the analysis of SSB-ssDNA interactions. ESI-MS was used to determine the rate of subunit exchange between unlabelled and uniformly 15N-labelled SSB, revealing a slow subunit exchange rate. Subunit exchange in a truncated version of SSB missing the last eight residues, SSBΔC8, proceeded markedly more rapidly in low salt, suggesting for the first time that the C-terminus plays a role in stabilising the SSB tetramer. This is proposed to occur via transient intersubunit interactions of the C-termini with the OB-folds from adjacent subunits, and may have implications for how other proteins access the SSB C-terminus.

ESI-MS analysis of SSB-ssDNA complexes formed under a variety of salt conditions allowed complexes corresponding to different SSB binding modes to be observed. Subunit exchange was dramatically inhibited when SSB was bound to ssDNA oligonucleotides, suggesting that DNAbinding locks the tetramer in place, even when only half the SSB subunits are DNA-bound. In addition, analysis of SSB transfer between discrete ssDNA molecules by ESI-MS revealed a dramatic preference for transfer to occur from an initial complex containing unoccupied SSB subunits, confirming the predictions from solution phase experiments which determined the mechanism of direct transfer via a ternary complex. This work showed, therefore, that SSBssDNA complexes observed by mass spectrometry offer an accurate reflection of the specific contacts formed in the different binding modes.

The protein clamp responsible for the processivity of E. coli DNA replication, β2, also contains a well-conserved site through which it interacts with a large number of binding partners. The β2 dimer operates by encircling and sliding along ssDNA, thus acting as a DNA tether for its binding partners. It contains an identical, primarily hydrophobic binding pocket on each of its subunits, enabling it to interact with a multiple polymerases and accessory subunits along with its primary contact with the replicative polymerase subunit, α. Recently in our laboratory, a previously undescribed interaction between β2 and the replicative exonuclease subunit, ε, was discovered.

The second aim of this thesis was to characterise the strength and several functional aspects of the newly identified ε-β2 interaction: particularly whether the interaction is mediated thorough the common protein binding cleft on the clamp. This required the creation of β2 dimers in which the putative ε-binding cleft was disrupted in either one or both subunits. Attempts to create such dimers by fusing both subunits into a single gene product were unsuccessful. Instead, the subunit exchange properties of the β2 dimer in various salt concentrations were determined by ESI-MS, and this information was used to create and purify hybrid dimers containing a single, intact binding site. Functional replication assays using these dimers showed that ε-dependent rolling circle replication reactions were dependent on the presence of both intact binding clefts in the β2 dimer, indicating that the ε-β2 interaction occupies the predicted binding pocket on β2, and occurs concurrently with the β2-polymerase interaction. These assays also suggested that the role played by the ε-β2 interaction during DNA replication is to stabilise the replication fork. Surface plasmon resonance measurements of the affinity of the interaction between β2 and a peptide corresponding to the proposed clamp binding motif from ε showed that the interaction was weak (KD ~200 μM). Thus, the ε-β2 interaction, whilst transient alone, likely gains its significance as part of a complex network of protein-protein interactions at the replication fork.