Mechanisms of replication fork-associated versus post-replicative bypass of abasic sites

• Applicant: Professorin Helle Ulrich, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 548574498

Management of DNA replication stress is an important quality control measure that ensures cellular homeostasis as well as faithful transmission of an organism’s genetic information to the next generation. Several alternative pathways are available for the bypass of replication-blocking DNA lesions, either in association with the replication fork or in a postreplicative mode. Since they operate with varying degrees of fidelity, the choice between these pathways is critical for controlling the level of mutagenesis during replication of damaged DNA. However, we have limited knowledge regarding the timing of damage bypass relative to replisome progression through the DNA lesion and how pathway choice affects and is coordinated with overall replication fork progression. Recently, we have developed a microscopy-based assay for real-time, locus-specific monitoring of replication rates in budding yeast. Our unpublished data indicate that this system can be harnessed to detect and quantify the degree of replication fork stalling and the formation of daughter-strand gaps, i.e., discontinuities in the newly synthesized DNA associated with postreplicative damage processing. In addition, we have developed a method for the site-specific enzymatic introduction of DNA lesions into the yeast genome. We now plan to combine these tools to investigate the temporal and spatial coordination between replisome progression through DNA damage and the activation of damage bypass pathways. We will focus our analysis on abasic sites, which are among the most abundant types of endogenous lesions and, at the same time, elicit a complex response in the cell due to their reactivity and their potential to form derivatives such as DNA-protein crosslinks and strand breaks. We will first validate our system by determining the load of lesions and their distribution using quantitative genome-wide mapping technology. We will then use systematic variations in the genetic background of our experimental strains to query the relative contributions of the various damage bypass and DNA repair pathways to fork rates, gap formation, and damage processing. Our analysis will, for the first time, provide quantitative real-time insight into the replication of a spatially defined damaged genomic region in live cells. In this manner, we expect to advance our fundamental understanding of the events and factors that orchestrate the processing of an abundant, yet potentially highly dangerous lesion at and behind the replication fork.

• DFG Programme: Research Grants
• International Connection: Israel
• International Co-Applicant: Professor Amir Aharoni, Ph.D.