Real time measurement of replication fork progression through site specific DNA damage

DNA damage and repair is the focus of extensive studies that utilize a variety of DNA damage reagents for generating global DNA damage throughout the chromosomes. To generate a highly tunable and controlled system for the study of replication through damaged DNA, we set out to create an experimental system capable of inflicting site-specific DNA damage in living eukaryotic cells, during a specific cell cycle phase and with a tuneable degree of severity. We utilize engineered enzymes that create specific DNA lesions, including DNA glycosylase and deaminases, fused to a dead Cas9 (dCas9) to target the fusion protein to a specific genomic location using a guide RNA molecule. To tune damage severity, we introduced a synthetic array, composed of multiple repeats of a 25bp gRNA target and 20bp random sequence spacer at a specific site of the yeast genome. Following galactose induced expression, multiple copies of the dCas9-enzyme-gRNA complex bind the target array and generate a site specific DNA damage. To eliminate continues damage during replication, dCas9-enzyme is degraded using Auxin induced degradation (AID) system by addition Indole-3-acetic acid (IAA) to the medium. This system is employed together with a bacterial fluorescent repressor-operator system (FROS) in Saccharomyces cerevisiae (budding yeast) allowing measurements of DNA replication through different types of damaged DNA using time-lapse fluorescent microscopy.