Mechanistic Insights into Single-Strand Break Repair Within Chromatin

Project Summary With cells constantly encountering DNA damaging agents, mechanisms for prompt DNA repair are crucial for maintaining genomic stability. Aberrations and mutations in the proteins involved these processes are closely associated with a plethora of diseases, including cancer and neurological disorders. Among various

types of DNA damage, single-strand breaks (SSBs) are the most prevalent, occurring at a rate of 55,000 per cell daily. Understanding the repair of SSBs within chromatin through the base excision repair (BER) mechanism is the primary focus of the proposed research project. The proposed research project primarily aims to unravel

the complexities of SSB repair within chromatin through the BER mechanism. This multifaceted pathway involves poly-ADP-ribosylation (PARylation) and histone acetylation, both closely associated with DNA repair. PARP1, the most abundant member of the PARP family, is the enzyme that confers poly-ADP-ribosyl groups to

histones upon detection of SSBs. This PARylation then recruits and activates various repair proteins like DNA Ligase III (LIG3) and Polynucleotide Kinase 3'-Phosphatase (PNKP). PNKP has dual functions in the process, particularly for SSBs induced by radioactivity, by adding a phosphate group to the 5'-end of DNA breaks and

removing one from the 3'-end. This prepares the DNA ends for the action of DNA ligases, such as LIG3, thereby playing a vital role in DNA repair mechanisms. This proposal aims to understand how three DNA repair proteins—PARP1, LIG3, and PNKP—recognize and repair SSBs within the context of nucleosomes and to

explore the interplay between PARylation and acetylation of histones in these processes. We will investigate how PARP1 identifies and recognizes SSBs within nucleosomal contexts, a critical phase in the DNA damage repair process. Additionally, we will explore the roles of enzymes LIG3 and PNKP in repairing SSBs within

nucleosomal structures. To achieve these goals, we will map the accessibility of SSB sites within nucleosomes using DNA libraries and next-generation sequencing, aiming to optimize the positioning of an SSB for facilitating the formation of nucleosome complexes with the DNA repair proteins. Subsequently, we will employ biophysical

tools and functional assays to characterize these interactions and functions, both in the presence and absence of histone modifications. Finally, we will leverage state-of-the-art NMR techniques and cryo-EM to obtain detailed structural and dynamic information about the nucleosome when complexed with PARP1 (or its zinc finger

domains), LIG3, and PNKP. The long-term goal of this research is to provide an enhanced fundamental understanding of BER mechanisms within nucleosomal contexts, laying a molecular foundation for the design of drugs and therapeutics that can beneficially modulate these mechanisms in various disease states.