Title: DNA Damage Tolerance Laboratory
Funder: Cancer Research UK

Abstract: My lab is interested in understanding how post-translational protein modifications by members of the ubiquitin family, such as ubiquitin and SUMO, contribute to mutagenesis and the maintenance of genome stability, in particular in the context of lesion bypass during DNA replication. DNA is susceptible to a variety of insults from exogenous and endogenous sources. In contrast to DNA repair systems, which usually rely on the excision and subsequent re-synthesis of the damaged region to restore the original sequence information, DNA damage tolerance mechanisms allow the bypass of lesions without their actual removal. They ensure the completion of DNA replication on damaged templates and are therefore essential for survival of a cell in the presence of genotoxic agents. As lesion bypass is often associated with damage-induced mutations, however, its activity needs to be tightly controlled. Our research aims at understanding the mechanisms and signals by which the ubiquitin system promotes damage tolerance and limits the accumulation of unwanted mutations. Replicative bypass of DNA lesions, also called postreplication repair, is controlled via ubiquitylation of the replication factor PCNA in response to DNA damage. Whereas monoubiquitylation activates translesion synthesis by damage-tolerant DNA polymerases, polyubiquitylation via lysine 63-linked chains is required for an error-free pathway of damage avoidance possibly involving template switching. However, the way in which PCNA polyubiquitylation activates the damage avoidance pathway, as well as its mechanistic details, are poorly understood. Over the past year, research in our lab has therefore concentrated on the functions and interaction partners of polyubiquitylated PCNA. Three non-exclusive models can be envisioned for the function of PCNA polyubiquitylation in damage bypass: (A) the polyubiquitin chains could target PCNA for degradation by the proteasome in order to prepare the stalled primer terminus for template switching; (B) extension of the monoubiquitin moiety to a polyubiquitin chain on PCNA might block the access of damage-tolerant polymerases, thereby allowing the alternative pathway of template switching to proceed; or (C) polyubiquitylation of PCNA might facilitate the association of a downstream effector protein that induces the error-free pathway. Our previous work had revealed that the ubiquitin chains attached to PCNA are rarely longer than three or four units, and that polyubiquitylated PCNA is not a substrate of the proteasome, thus excluding model A. Complementation studies with mimics of polyubiquitylated PCNA involving linear fusions of ubiquitin to PCNA had indicated that sterically blocking the N-terminus of the ubiquitin that is attached to PCNA may indeed prevent translesion synthesis, thus lending some support to model B. However, our recent data suggest that a ubiquitin chain of 3 or more units is more effective at template switching than a chain that is limited to 2 moieties. Hence, it is likely that the function of polyubiquitylated PCNA goes beyond the inhibition of translesion synthesis, suggesting a contribution of model C. Potential downstream effectors of polyubiquitylated PCNA should exhibit a basal affinity for native PCNA, but their interaction with the modified form should be enhanced by means of ubiquitin-binding domains. One such candidate protein is the AAA+ ATPase Mgs1 (or its human homolog, WRNIP1), which is known to interact with PCNA and harbours a ubiquitin- binding zinc finger (UBZ) domain of poorly defined function. Mgs1 contributes to genome stability both during undisturbed replication and in response to DNA damage. Deletion of the gene, but also overexpression, causes hyperrecombination, and the protein had been suggested to exert a negative impact on the replicative DNA polymerase δ. By means of genetic analysis, we were able to show that the UBZ domain exclusively mediates the damage-related functions of Mgs1, and that these are fully dependent on the ubiquitylation of PCNA. We found that the protein indeed preferentially interacts with mono- and even more so with polyubiquitylated PCNA in a UBZ-dependent fashion. As a consequence, the UBZ domain promotes the recruitment of Mgs1 to replication intermediates under conditions of DNA damage and replication fork stalling, i.e. when PCNA ubiquitylation can be observed . Three-hybrid analysis indicates that Mgs1 interferes with the physical interactions between PCNA and polymerase δ, which suggests that the protein might contribute to damage bypass by facilitating the exchange of PCNA binding partners in preparation for translesion synthesis or template switching. Hence, our findings have identified Mgs1 as a downstream effector of ubiquitylated PCNA that might contribute to damage bypass in a regulatory manner. However, it is also evident that the action of Mgs1 alone cannot fully explain the error-free pathway. Therefore, the function of PCNA polyubiquitylation is most likely not limited to the recruitment of a single effector protein, but plays a more diverse role in signalling than anticipated.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS