Abstract 2017 A Revised Mechanism of Hydroxyurea Induced Replication Stress

Hydroxyurea (HU) is commonly used in yeast to induce replicative stress through the inhibition of dNTP synthesis, leading to activation of the S-phase checkpoint. The mechanism by which this is accomplished involves disrupting the function of the enzyme Ribonucleotide Reductase (RNR) through quenching of a catalytic tyrosyl radical. However, research into archaeal systems that have RNR enzymes which do not possess a tyrosyl radical have still shown susceptibility to HU inhibition. HU has also been shown to only partially deplete dNTP levels, but did not deplete them completely. This presents a need for further exploration into other potential mechanisms through which HU may be acting in disrupting the cell cycle. A potential alternative source for the observed replicative stress can be found in reactive oxygen species (ROS). HU is known to produce ROS, such as H2O2, within yeast. These ROS have the potential to disrupt replication by acting on special [4Fe-4S] clusters found in the catalytic domains of several eukaryotic replicative polymerases. These include polymerase α, the lagging strand polymerase δ, and the leading strand polymerase ε. Here, we show that the iron sulfur region is implicated in both polymerase complex stability and DNA binding. As such, disruption of the region has the potential for intense replicative stress through inhibition of polymerase activity. Given these unintended consequences of HU use, a need arises for alternative methods of studying replicative stress with more specificity. We also demonstrate a genetically implemented system in yeast that is able to produce replication stress without the unintended production of damaging ROS. This project was funded by the NIH (R35 GM147105)