Investigation of the Role of Iron-Sulfur Clusters in Polymerase Alpha Primase

Abstract

Faithful duplication of DNA lies at the center of DNA replication, defects in which can lead to cancer and other diseases of genomic instability. Generating copies of the two stands of the DNA duplex begins with synthesis of a primer, which is carried out by the heterotetrameric complex of DNA primase and DNA polymerase α (pol α), termed pol-prim. Primase builds the initial 7-12 nucleotide RNA primer, which is then handed off to pol α for extension by ~20 additional DNA nucleotides. Despite years of study, the mechanisms behind primer initiation, extension and handoff remain enigmatic. This dissertation investigates the role of the [4Fe-4S] clusters in pol-prim in priming and the hypothesis that the [4Fe-4S] clusters are involved in facilitating primer handoff. Here, I show in vitro that the [4Fe-4S] cluster in primase acts as a redox switch that leads to altered affinity of primase for DNA. This redox switch can be impaired in primase mutants. Biochemical assays with these primase mutants suggest that the redox switch may play a role in primase initiation and handoff. Furthermore, I provide evidence that the redox switch in primase is essential for viability in yeast and that pol α contains a [4Fe-4S] cluster. I propose that these results reflect the participation of pol-prim [4Fe-4S] clusters in regulation of primer handoff during replication and suggest that these [4Fe-4S] clusters play a larger role at the replication fork.