Conflicts between DNA replication fork progression and transcriptional regulation

All organisms must duplicate their genetic material so that it can be passed from one generation to the next. However, this process occurs in a crowded environment since other equally important cellular functions must occur at the same time as DNA replication. As a result, genome duplication must occur even though the DNA is coated with many DNA binding proteins. Little is known of the effects of these DNA binding proteins upon replication even though there is much indirect evidence that such collisions lead to mutations in the DNA. We aim to use the replication machinery from a bacterium to analyse the effects of proteins bound to the DNA upon replication. We will measure the probability of DNA binding proteins halting the replication machinery, and will characterise the properties of protein-DNA complexes which lead to blockage of replication. However, cells clearly do have the ability to replicate their genomes even though the DNA is coated in a panoply of proteins. We will therefore search for enzymes that can help the replication machinery to move through DNA-protein complexes. These experiments will help us understand how all cells duplicate their genomes in a complex protein-rich environment. They may also cast light on how cells minimise the chances of their replication machinery stalling and causing potentially harmful mutations.

Given the central role played by DNA replication in all forms of life, we know surprisingly little about this complex process. Most information has been gleaned from studies of replication in E. coli and its associated bacteriophages. However, these studies have centred largely on the mechanics of duplicating naked DNA templates in vitro. The situation in vivo is likely to be very different with a huge variety of proteins coating both prokaryotic and eukaryotic genomes. These DNA-bound proteins range from those associated with genome packaging to more sequence-specific proteins such as transcription factors. However, little is known about how cells duplicate protein-encrusted DNA templates. Indirect evidence suggests that proteins bound ahead of the advancing replication machinery can present blocks to replication and, as a consequence, may be associated with potentially harmful genome rearrangements. We aim to use an in vitro DNA replication system developed using purified enzymes from E. coli to address directly the barriers presented by DNA binding proteins to the replication machinery. Preliminary data suggests that transcription factors bound to the template DNA lead to stalling of replication forks in vitro. We will characterise the probability of transcription factors bound to their cognate DNA sequences of stalling the replication machinery, and determine the biophysical requirements for replication blockage. These data will shed light on the probability of replication forks stalling at DNA-protein complexes found in vivo and will provide a first glimpse of the hurdles which all cells must face in duplicating their genetic material. However, cells do achieve genome duplication in a protein-rich environment. This implies that accessory enzymes are needed in vivo to help replication progression through protein-DNA complexes. We will attempt to identify these accessory factors using our in vitro assay system. We aim to detect enzyme activities which facilitate replication fork progression through protein-DNA roadblocks by screening both candidate purified enzymes and partially purified E. coli cell-free extracts. Identification of such factors will help understand how all cells overcome potential blocks to genome duplication. These proposed studies will provide insight into the biophysical barriers that replication forks must overcome in all organisms, and may also shed light on the molecular basis of the generation of replication fork pause sites and hotspots of recombination.