DNA Damage Tolerance Laboratory

Previous and current research
Ubiquitin and SUMO are members of a conserved family of proteins that exert their functions by being covalently attached to intracellular substrate proteins. Ubiquitin is best known for targeting its substrates to the 26S proteasome, but it mediates many non-proteolytic functions, mainly by modulating protein-protein interactions.

Similarly, SUMO controls a variety of biological processes by changing the properties of its substrates. Both ubiquitin and SUMO play a prominent role in the maintenance of genome stability, and our lab is interested in elucidating the mechanisms by which the two modifiers affect DNA replication, repair and mutagenesis.

An important focus of our lab is the investigation of DNA damage tolerance, a highly conserved mechanism that protects cells from the harmful effects of genotoxic agents by allowing the replication machinery to pass over DNA lesions. Although beneficial in terms of promoting cell survival, bypass of DNA damage can result in unwanted mutations that may ultimately lead to genetic instability and cancer. The activity of lesion bypass is carefully controlled through the modification of PCNA, an essential processivity factor for DNA polymerases, by monoubiquitin, polyubiquitin chains and SUMO.

Using budding yeast as a model organism, we found that monoubiquitylation of PCNA activates error-prone translesion synthesis by specialised, damage-tolerant DNA polymerases. Modification of PCNA by the ubiquitin-related protein SUMO, on the other hand, recruits a helicase to the replication fork that prevents unscheduled recombination and allows ubiquitin-dependent damage bypass to proceed. Our findings have given insight into the mechanisms by which ubiquitin and SUMO affect the properties of their common target, PCNA, and into the cellular signals that trigger the appropriate modification in the cell. They suggest the how a cooperation between the ubiquitin and SUMO system, by controlling the accuracy of replication and damage tolerance, can contribute to overall genome stability.

Future projects
We are now engaged in the elucidation of the molecular mechanisms by which PCNA polyubiquitylation controls lesion bypass. By identifying and characterising factors that specifically interact with polyubiquitylated PCNA, we hope to gain insight into the downstream effects of this modification, but we are also interested in studying the recognition of the polyubiquitin signal by dedicated interaction domains in a wider context.

We are using genetic approaches to investigate the impact of DNA damage tolerance in a physiological context by analysing the consequences of PCNA modifications on components of the checkpoint response, mismatch repair and chromatin assembly. We have reconstituted the in vitro modification of PCNA with ubiquitin and SUMO in a fully recombinant system and are now investigating the conjugation mechanisms, the cooperative and competitive relationships between the individual conjugation factors and their interactions with the substrate and with DNA.

Finally, we are analysing a range of newly identified chromatin-associated SUMO targets for their contribution to genome stability. We hope that our efforts will contribute to a better understanding of how a cell uses posttranslational protein modifiers like ubiquitin and SUMO to maintain an appropriate balance between DNA replication, repair and mutagenesis. While we primarily use budding yeast as a genetically tractable model system, we are pursuing some questions in mammalian cells and have initiated more biochemically oriented projects using Xenopus laevis egg extracts.