Recombination and the clearance of replicative blocks - to bypass or not to bypass?

The ability of a cell to form two new daughter cells allows organisms to grow and reproduce. This process requires the copying of vast amounts of DNA so that each daughter receives an accurate copy of all the genetic information required for survival. Because of the importance of generating accurate copies of the DNA, organisms have evolved very complex DNA replication machines that reduce the chances of mistakes being made. Unfortunately, we now know that many obstacles are encountered by these replication machines that, if not overcome, can cause mistakes in the copying process. Such mistakes can lead to errors in the genetic information that can have fatal consequences.

Proteins that coat the DNA form frequent obstacles to the DNA copying machinery. These proteins are essential in all organisms for maintaining, reading and packing the genetic information, and so cannot be avoided. Although the DNA replication machinery can successfully push off most of these proteins from the DNA to be copied, the sheer number of proteins bound to the DNA mean that occasionally the copying process is stopped in its tracks. However, a specific class of enzymes can repair and restart broken down DNA replication machines. It is clear that these recombination enzymes can help to copy DNA that is bound by proteins but how they do so remains unclear. One proposal is that if a DNA replication machine becomes blocked by a protein then recombination enzymes can restart DNA replication on the other side of the protein block. This pathway would skip over the block, allowing copying to continue, but would also result in genes near the protein block not being copied. Consequently the involvement of recombination enzymes could be seen as harmful, resulting in failure to copy all the genetic information needed by the two daughter cells to survive. Alternatively, we have proposed that recombination enzymes might simply restart DNA replication near to where it initially came to a halt. This might give the DNA replication machinery a second chance to push the blocking protein off the DNA and continue to copy all of the genetic information. Such a process might therefore provide a mechanism to ensure accurate copying of DNA coated with proteins.

We will use the model bacterium E. coli to determine the roles of recombination enzymes in copying DNA coated with proteins. We know a great deal about the basic mechanisms of both DNA replication and recombination in E. coli, allowing us to analyse how these two very complicated processes interact. We will determine the mechanisms by which recombination enzymes can help DNA replication machines to move through protein blocks. We will also establish what dictates the balance between accurate and inaccurate recombination mechanisms to understand when such processes might generate potentially very harmful changes to the genetic material.

The conflict between the need to copy DNA and the need to have proteins bound to the DNA is one that all organisms must somehow resolve. This work will address therefore exactly what drives the accumulation of mutations within genes and what mechanisms help to minimise this accumulation. Acquisition of mutations in the genetic material is the driving force of evolution but such mutations are frequently harmful rather than beneficial and so must be kept in check. This is illustrated by the importance of mutations in the acquisition of human genetic disorders and the development of cancer. Understanding fundamental mechanisms of DNA replication and recombination in E. coli has greatly advanced our knowledge of genetic stability in more complex organisms such as ourselves. We are now in a position to use E. coli to address the interplay between these critical processes, an interplay that is central to understanding how genes are copied in as accurate a manner as possible.

High fidelity genome duplication demands highly processive DNA replication machines. But the complex intracellular environments in which genome duplication must occur present many challenges to continued replication fork movement. The cost of failure to surmount such challenges is high with replicative problems emerging as a key source of genome rearrangements in all organisms. A source of highly abundant, unavoidable barriers to replication are proteins bound to DNA. However, the mechanisms that underpin replication of protein-coated DNA are only just being uncovered. Recombination enzymes may provide one system that promotes fork movement through protein-DNA complexes. However, many models invoke bypass of nucleoprotein barriers by recombination, one consequence of which is gross chromosomal rearrangements. Thus recombination might be a pathological event that occurs only upon failure of other mechanisms to promote duplication of protein-bound DNA. However, our data suggests that recombination enzymes may play critical roles in the normal and accurate replication of protein-bound DNA. We aim to exploit the genetic and cell biological tools we have developed in E. coli, together with the detailed mechanistic information available in this organism, to study the interplay between recombination and replication. We will determine the relative contribution of recombination enzymes in promoting genome duplication and establish models of recombination-promoted replication in the face of protein-DNA barriers. We will also analyse the factors that dictate the balance between accurate versus inaccurate recombinational repair of blocked replication forks. These studies will provide insight into how all organisms employ recombination to overcome nucleoprotein replicative barriers, how these processes contribute to the maintenance of genome stability but also the circumstances under which genome instability is the outcome.

This proposal aims to delineate the roles played by recombination in maintaining accurate replication of DNA in the face of nucleoprotein complexes but also the circumstances under which occasional mistakes in these processes may lead to the generation of gross chromosomal rearrangements. The fundamental studies proposed here will provide therefore a mechanistic basis for understanding one potential source of mutations that may contribute to the development of genetic disease and also the accumulation of the many mutations now known to occur during carcinogenesis. Scientists and clinicians with interests in the roles of mutation in human and animal disease will therefore benefit from this research.

Pharmaceutical companies will be a second beneficiary of this work. DNA replication and DNA repair are both important targets for cytotoxic agents and knowledge gained from this proposal may underpin development of anti-cancer agents. Many current antimicrobial agents also target DNA replication and so this proposal may facilitate the development of novel agents for the treatment of bacterial and viral diseases. Additionally, many companies have developed diagnostic reagents concerning the contribution of genome instability to human health. Knowledge gained from this proposal may facilitate continued development of such diagnostic tools.

This proposal may also benefit biotechnology companies and others with interests in development of novel DNA modification tools. Enzymes involved in DNA recombination and repair, the central focus of this application, are employed in many molecular biology technologies. For example, RecA, the E. coli strand exchange protein, has been exploited for D-loop mutagenesis, facilitation of cDNA cloning, engineering of complex higher eukaryotic genomes and many other applications. Given that this proposal seeks to understand mechanisms of recombination, this work may underpin development of novel applications of recombination and repair enzymes in the manipulation of DNA molecules in vitro and modification of genomes in vivo.

Training of the research workers and students during this project will contribute to the generation of a scientifically-literate workforce. This will have direct benefits to the UK economy by enhancing international competitiveness in technologically-advanced industries and so will contribute to the economic well-being of the general public. The general public will also derive other benefits from this proposed work. Underpinning of the development of therapeutic and diagnostic reagents for human and animal disease and of the generation of novel antimicrobials will benefit the health and well-being of the UK population. This proposed research will also contribute to the public understanding of health care issues with respect to mutation and may clarify the confusion in the public's mind with respect to the links between diet, environment, mutation and cancer. More generally, this project is focused on DNA and genes, subjects that are relatively familiar to the general public. Researchers trained during this project will therefore be aided in engaging with the public on topics such as genetic testing, stem cells and the "nature versus nurture" debate.