High performance motoring - replication fork movement in a complex environment

DNA replication is essential to pass on the genetic blueprint from one generation to the next. Moreover, duplication of DNA must be done with the highest possible accuracy. Even small mistakes arising during the duplication process can lead to the development of genetic disease and cancers in humans. To minimise the possibility of such mistakes being made during DNA replication, all organisms have evolved complex biological machines to perform this process both speedily and accurately. However, recent work has shown that despite the best efforts of evolution, this DNA replication machinery does break down. If not restarted, these breakdowns lead to potentially catastrophic errors arising in the genetic blueprint. We have developed a system to monitor the breakdown of replication using the simple DNA replication apparatus of a bacterium. This current proposal aims to determine the features of replication machines that help minimise their breakdown. But when replication does break down, there are systems that attempt to restart the replication process. We will therefore also try and establish whether the repair systems interact with the replication machinery to help make the repair process more efficient. These studies therefore aim to characterise how organisms minimise the chances of DNA duplication going awry to try and avoid the disastrous consequences of defects arising within the genetic material.

The breakdown of DNA replication is emerging as a major cause of genome instability in all organisms. Whilst the reasons for these breakdowns have been obscure, our recent work has shown that proteins bound to the template DNA can be a major impediment to movement of the replication machinery of E. coli. Despite this, replication complexes do have some ability to translocate along protein-bound DNA. Furthermore, we have shown that if replication forks do become blocked at protein-DNA complexes, they retain the capacity to resume replication if the block is removed within a few minutes of the initial blockage. These findings suggest that replication complexes have evolved at least some capacity to duplicate protein-encrusted DNA. This capacity may be critical in minimising the frequency of replication breakdown and the associated risks of genome instability. However, the basis for this capacity remains unknown. We propose to identify features of the replication machine in E. coli that enhance the ability to proceed through protein-DNA roadblocks by utilising a biochemical reconstitution approach. We will also determine which features of the replisome are important in maintaining the ability of blocked replication forks to resume replication if the block can be removed. However, when forks do break down irreversibly, then replication must be restarted. Evidence is emerging that replication repair enzymes associate physically with components of the replication machinery. We will investigate whether such interactions do exist, and the functional consequences of such interactions for the efficiency of replication restart. These studies will identify properties of the E. coli replisome that facilitate genome duplication in a complex, protein-rich environment. Given that the genomes of all organisms are unavoidably coated with proteins, these experiments may highlight general features of replication fork propagation that are critical in minimising fork stalling, and thus genome instability, in all domains of life.