Structural biology of DNA damage response in chromatin

Summary/Abstract When DNA is damaged, cells activate a complex network of proteins that sense, signal, and initiate repair. This DNA damage response (DDR) also encompasses mechanisms for DNA damage tolerance, enabling cells to replicate damaged DNA. In eukaryotes, the DDR operates within chromatin, consisting of repeating nucleosomes where histone proteins and DNA serve as docking surfaces for DDR proteins. Central to signaling DNA double- strand breaks (DSBs) is the mammalian E3 ubiquitin ligase RNF168. RNF168 catalyzes the ubiquitylation of histone H2A and variant H2AX at DSB sites, regulating the recruitment of several DDR proteins to chromatin, including those involved in homologous recombination or homology-directed repair (HDR). Despite progress in understanding RNF168-mediated signaling, key gaps remain, particularly regarding the function and mechanisms of action of E3 ubiquitin ligase RAD18, one of the effector proteins of RNF168. The central hypothesis of this proposal is that downstream effectors of RNF168 integrate DNA damage repair with DNA damage tolerance, focusing primarily on the E3 ubiquitin ligase RAD18. RAD18 is unique in its involvement in both HDR and DNA damage tolerance pathways. The planned research will investigate the role of RAD18 in HDR and other repair pathways activated by replication stress using integrative structural biology, including single-particle cryo-electron microscopy (cryo-EM), NMR spectroscopy, X-ray crystallography, biophysical approaches, chemical biology, and collaborative cell biology. Specifically, the nucleosome association of RAD18 and of chromatin maintenance proteins recruited in a RAD18-dependent manner will be characterized. Additionally, mechanisms through which RAD18 and cognate E2 ubiquitin-conjugating enzyme RAD6 promote DNA damage tolerance via the mono-ubiquitylation of DNA sliding clamp PCNA will be examined. The research will also explore a mechanism involving RAD18-RAD6 that integrates elements of both DSB repair and DNA damage tolerance through PCNA-ubiquitylation-directed homologous recombination. Overall, this work will contribute fundamental knowledge that enhances the understanding of DNA damage repair and tolerance mechanisms, including how they are integrated. Given the importance of the DNA damage response in maintaining genomic stability and preventing diseases such as cancer and neurological disorders, these studies are expected to yield new findings that will have long-term benefits for human health. During this research, it is probable that unexpected and intriguing new questions will arise, and some of these may be addressed under the flexibility afforded by the MIRA mechanism.