As part of a 4 year KWF funded project that concluded in 2006, we made several significant finding in the mechanism of a distinct pathway that promotes the tolerance of DNA damage during its replication phase. Previous knowledge from lower organisms suggested the requirement for enzymes capable of constructing a chain of ubiquitin molecules linked in a specific manner. We used a novel approach to disrupt the formation of these ubiquitin chains in human cells and found that this caused a significant increase in mutations after exposure to UV light. Several lines of evidence implicate a family of error-prone enzymes, called translesion synthesis polymerases, in the formation of these mutations. Furthermore, we provide the first evidence suggesting that proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, is the relevant target of these chains in human cells. This study was published in the prestigious Public Library of Science and received attention in newspaper articles in the Netherlands and Canada. (Chiu et al., PLoS Genetics. 2(7):e116)
We extended this study to investigate the mutagenic effects of the class of environmental carcinogen called polycyclic aromatic hydrocarbons. We hypothesized that disrupting the formation of K63-polyUb chains inhibits damage avoidance and favors error-prone repair involving low-fidelity polymerases (e.g. POLh), causing increased BPDE-induced mutagenicity. Together with Prof. Frederik-Jan van Schooten of NUTRIM, we demonstrated that formation of K63-polyUb chains protects BPDE-exposed human cells against translesion synthesis-mediated mutagenesis. These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by contributing to genomic stability. (Langie et al., DNA Repair (accepted))
In a manner analogous to phosphorylation, protein modification with polyubiquitin chains is emerging as an important modifier of many signalling pathways including DNA repair. Our understanding of this process in cellular signalling is limited because we lack knowledge of involved substrates. In collaboration with Prof. Edwin de Pauw at the University of Liege, we developed a novel method to isolate linkage specific ubiquitin substrates. Mass spectrometry based determination of K63-linked ubiquitination substrates revealed several known targets including L27a, EF1A1 and NMD3. In addition we identified 14-3-3Z/S as a new target of these novel chains. This work has been submitted for publication in 2006.
Selected publication
Chiu, R.K., Brun, J., Ramaekers, C., Theys, J., Weng, L., Lambin, P., Gray, D.A, and Wouters, B.G.
Lysine63 poly-ubiquitination protects cells against mutations induced by DNA lesion bypass polymerases.
PLoS Genetics (2006). 2(7):e116.
Figure: We have developed a system to selectively block specific types of ubiquitin chains in human cells. Disruption of ubiquitin chains like via lysine 63 leads to a dramatic increase in mutation frequency which corresponds to an increased recruitment of error prone DNA polymerases (demonstrated by foci formation). These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by contributing to genomic stability.
Research groupProf. Bradly G. Wouters, projectleader
Dr. Roland Chiu
Prof. Philippe Lambin
Dr. Jan Theys
Post-doctoral fellows
Dr. Christelle Vreuls
PhD students
Chantal Ramaekers
Technicians
Kim Paesmans
Students
Elena Bardina