An investigation of DNA repair processes during DNA replication using the archaeon, Sulfolobus, as a model organism.

Mutations in DNA are responsible for a number of medical diseases including cancers. The aim of this study is further our understanding of how cells prevent and repair DNA damage during DNA replication, the process when DNA is copied prior to cell division. The intricacy of the mechanisms involved in human and animal cells can complicate direct experimental observation, and for this reason we often use ‘simple‘ model organisms to gain further insight into these processes. In this study, I intend to examine new aspects of DNA repair using a single-celled model organism that thrives in harsh environmental conditions at temperatures of up to 80 C. This organism, called Sulfolobus, belongs to an ancient superfamily of microorganisms called archaea that are genetically distinct from bacteria. In fact, the archaeal apparatus for DNA replication and repair is more closely related to the machineries found in animal cells rather than those present in bacteria. Sulfolobus is also easy and inexpensive to cultivate, and the robustness of the heat-stable proteins isolated from these organisms facilitates experimental investigation. Therefore, Sulfolobus is an ideal organism in which to study the basic processes of replication-associated DNA repair.

In all organisms, repair of DNA damage is critical to maintaining genomic integrity and therefore viability. Indeed, the loss of chromosomal stability is coincident with the development of many cancer related diseases (1, 2). The process of replication fork restart is central to the maintenance of normal genetic equilibrium (3). Illegitimate recombination and genomic instability can ensue following the defective repair of stalled replication forks. Unfortunately, the intricacy of replication restart pathways in metazoan organisms complicates direct experimental observation. I propose to elucidate the mechanisms of replication fork restart by taking advantage of the experimental tractability of the model organism Sulfolobus. By investigating the clear archaeal orthologues of eukaryotic repair proteins, I am optimistic that the general themes observed in this study will aid our comprehension of eukaryotic DNA replication associated repair processes.

The overall objective of this investigation is to understand how replication forks are stalled and restarted in the hyperthermophilic archaeon Sulfolobus, and relate the findings to eukaryotic systems. Specifically, I aim to improve our understanding of the macromolecular assembly of higher-order structures at stalled replication forks. Biochemical, structural and genetic approaches will be adopted to examine the interactions that underlie these critical fork restart mechanisms. Initially, yeast two-hybrid and co-immunoprecipitation approaches, using candidate repair proteins that have been implicated in restart processes, will be performed to identify novel proteins and complexes associated with replication fork restart. Subsequently, the DNA binding preferences and metabolism of these candidate restart/repair complexes will be investigated by biochemical assays, using synthetic DNA substrates mimicking replication forks and repair-associated structures. Collaborations will be developed to examine the structural basis of the most interesting interactions. Finally, the in vivo significance of these associations will be analysed in Sulfolobus cells using genetic approaches involving the generation of domain specific mutations of the candidate proteins. Ultimately, I am confident that this study will provide valuable insights into the structure and function of the protein complexes involved in eukaryotic replication fork restart mechanisms.

1. Thompson, L. H., and D. Schild. 2002. Recombinational DNA repair and human disease. Mutat Res 509:49-78.
2. Hickson, I. D. 2003. RecQ helicases: caretakers of the genome. Nat Rev Cancer 3:169-78
3. Branzei, D., and M. Foiani. 2007. Interplay of replication checkpoints and repair proteins at stalled replication forks. DNA Repair (Amst).