Helle Ulrich

Function of ubiquitin and SUMO in the maintenance of genome stability

The main focus of our lab is to understand how a cell manages to replicate its genome in the presence of DNA-damaging agents. Unlike DNA repair systems that process lesions in double-stranded DNA, damage tolerance mechanisms operate on single-stranded DNA and enable the replication machinery to bypass sites of damage without actually removing the lesion. Although they contribute significantly to survival, the activity of damage bypass needs to be carefully controlled, as the process of replication past a lesion is often inherently inaccurate and may lead to unwanted, damage-induced mutations in the genome.

Our lab is studying the mechanisms and the regulation of DNA damage tolerance using a combination of genetics, molecular biology and biochemistry. We use primarily budding yeast as a genetically tractable model organism, but we have also initiated biochemically oriented projects using the Xenopus laevis egg extract system.

Central to the coordination of DNA replication with lesion bypass and repair is PCNA, the processivity clamp of replicative DNA polymerases and interaction platform for a wide range of other DNA-processing enzymes. The range of PCNA signaling is considerably diversified through posttranslational modifications of the clamp by ubiquitin and the ubiquitin-like modifier SUMO. Previous work by our lab ^1-5 and others has established that the monoubiquitylation of PCNA activates damage-tolerant polymerases, specialised enzymes that can accommodate damaged templates in their active sites and thus contribute to the mutagenic bypass of lesions.

In contrast, polyubiquitylation allows lesion bypass in an alternative, error-free manner that is mechanistically not well understood. Finally, we found that SUMO modification of PCNA acts as a safeguard mechanism during S phase and prevents unscheduled recombination events at the replication fork. Thus, both ubiquitin and SUMO affect not only the efficiency, but also to the accuracy of replication.

A range of projects addressing the function of ubiquitin and SUMO in the maintenance of genome stability is available to prospective PhD students. In a biochemical approach, the student would investigate the interactions of the relevant ubiquitin and SUMO conjugation enzymes with the components of the replication machinery and with DNA, with the aim of reconstituting the DNA damage bypass reaction in vitro. This approach is expected to give insight into how the relevant modifying enzymes are recruited to stalled replication forks and how the various different modifications of PCNA - monoubiquitination, polyubiquitination and SUMOylation - are coordinated and controlled.

As an alternative project, the relevance of ubiquitin and SUMO for DNA damage tolerance could be addressed in vivo, using technology such as chromatin immunoprecipitation and DNA fiber analysis for determining the association of relevant proteins with DNA and measuring the consequences of replication stress or DNA damage on replication fork movement.

Finally, our lab has identified a number of new SUMO and ubiquitin targets and/or effector proteins relevant to DNA replication, repair and chromosome segregation in yeast and Xenopus laevis, which provide a variety of new project options for investigating how the two posttranslational protein modifications contribute to genome maintenance.

References

1. Stelter P and Ulrich HD. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 2003; 425: 188-191.
2. Ulrich HD. Trends Cell Biol 2005; 15: 525-532.
3. Papouli E, Chen S, Davies AA, Huttner D, Krejci L, Sung P and Ulrich HD. Mutual interactions between the SUMO and ubiquitin systems: a plea of no contest. Mol Cell 2005; 19: 123-133.
4. Sarkar S, Davies AA, Ulrich HD and McHugh PJ. DNA interstrand crosslink repair during G1 involves nucleotide excision repair and DNA polymerase zeta. EMBO J 2006; 25: 1285-1294.
5. Guo C, Sonoda E, Tang TS, Parker JL, Bielen AB, Takeda S, Ulrich HD and Friedberg EC. REV1 protein interacts with PCNA: significance of the REV1 BRCT domain in vitro and in vivo. Mol Cell 2006; 23: 265-271.