Replication through structure-prone DNA – mechanism and impact on genome stability

This proposal is aimed at understanding how repetitive and structure-prone sequences are replicated accurately. Repetitive DNA comprises over half of the human genome. Repeat instability due to unusual DNA secondary structures causes many neurodegenerative diseases and is a diagnostic and prognostic cancer marker. While genetic studies revealed that repeats impair DNA replication and that replication can induce repeat instability, mechanistic insight is lacking. It is therefore crucial to understand how repeats are accurately replicated in normal tissues and how they become unstable in disease. First, I will determine the nature of replication-dependent DNA structures. Building on my recent advances in sequence-specific in vitro replication, I will delineate the effects of DNA secondary structures on replication dynamics, including DNA unwinding, DNA synthesis, Okazaki fragment maturation and replisome stability. Finally, I will discover factors that enable faithful replication of structure-prone DNA using a combination of biochemistry, candidate genetics and proteomics. As a long-term goal, I aim to elucidate the interplay between replication and repair pathways, such as mismatch repair. Altogether, these aims will define the relationship between DNA sequence, structure and replication and may lead to identification of novel factors that modulate the formation and resolution of toxic DNA structures.