Functions of replication clamp loading and modification in DNA damage tolerance

• Applicant: Professorin Helle Ulrich, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 493154786

Faithful and efficient processing of DNA damage during genome duplication is essential to prevent mutagenesis, chromosomal rearrangements and the development of cancer. The pathway of DNA damage bypass plays an important role in the protection of the genome from replication stress, but it can also contribute to genomic instability by activating DNA damage-induced mutagenesis. Insight into its regulation is therefore key to understanding the mechanisms that prevent or promote malignancies. Here we propose to explore the dynamics of a central DNA replication factor, the processivity clamp PCNA, and the implications for the efficiency and accuracy of DNA damage bypass. Modification of PCNA by the posttranslational modifiers, ubiquitin and SUMO, is well known to regulate the choice between accurate and mutagenic pathways of DNA damage bypass; however, we still understand very little about the interrelations between these modifications and the factors that activate and inactivate PCNA by mediating its loading onto DNA and its unloading from the chromatin. A series of alternative clamp loaders has been genetically associated with genome maintenance. We will now examine how these loaders control the residence of PCNA at stalled forks and postreplicative daughter-strand gaps, respectively, how they are affected by PCNA modifications and how they influence the genome-wide distribution of DNA damage bypass activity. Further, we will address the question of how the PCNA modification pattern, i.e., the distribution of ubiquitin and SUMO over the three subunits of the homotrimeric clamp, impinges on DNA damage bypass and the interactions with the loaders as well as important mediators of the pathway, such as the ubiquitin ligase Rad5 and the damage-tolerant DNA polymerases involved in translesion synthesis.We will pursue these questions by means of a combination of genetic and cell biological assays in the model organism Saccharomyces cerevisiae, where a set of dedicated tools, such as regulable promoters and orthogonal degron systems, will allow for a rapid and precise manipulation of protein levels over the course of a cell cycle. In addition, an engineered version of PCNA will serve to dissect the modification pattern of the clamp. We will complement our genetic analysis with next-generation sequencing experiments for a genomic view of DNA damage bypass and with biochemical assays addressing the mechanism of PCNA modification. The experiments described here will provide insight into the basic mechanisms at work in this universally conserved pathway of genome maintenance and may provide a basis for future research aimed at inhibiting DNA replication in tumor cells or overcoming the mutagenic effects associated with the tolerance of DNA lesions.

• DFG Programme: Research Grants
• International Connection: Israel
• International Co-Applicant: Professor Dr. Martin Kupiec