Elucidating the role of the iron-sulfur cluster in the nuclease/helicase Dna2

The genetic blueprint of all living organisms, whether unicellular (e.g. bacteria) or multicellular (e.g. human) is encoded by its genome. One of the most fundamental challenges of the cell is to accurately copy and transmit its genome to daughter cells. In addition, during their life cycle cells are often exposed to many forms of DNA replication stress and DNA damage, and combat this via the complex network of interconnected pathways, named the DNA damage response (DDR). If this process goes awry, genomic instability develops and, depending on specific mechanisms involved, presents in various forms and represents a major hallmark of cancer. Over the years, a number of critical proteins in DNA replication and repair have been shown to coordinate an FeS cluster (i.e. DNA2, DNA primase, Pol, Pol, Pol, MUTYH, XPD, RTEL1, FANCJ, and ChlR1). These findings were rather surprising, given that upon FeS cluster oxidation, free iron atoms can generate dangerous reactive oxygen species that may interfere with DNA integrity and lead to genomic instability. So far, the function of FeS clusters in these proteins is largely unknown. Since all known FeS cluster proteins interact with MMS19/MIP18, core members of the Cytoplasmic Iron-sulfur Assembly (CIA) machinery, we reasoned that new FeS proteins may be amongst the interaction partners of MMS19/MIP18, previously identified by mass spectrometry. In this study we focused on Replication Factor C subunit 5 (RFC5) since it not only associates with MMS19/MIP18, but also displays synthetic lethality with mms19 in yeast, a feature observed for multiple FeS proteins. Using coimmunoprecipitation interaction studies with the CIA machinery and 55Fe incorporation assays, our data show that while RFC5 interacts with both MMS19 and MIP18, it is unlikely to coordinate an FeS cluster. Additional studies will be required to identify the functional relevance of RFC5’s interaction with the CIA machinery. In our second study, we aimed to further characterize the role of the FeS cluster in the nuclease/helicase Dna2, a known FeS cluster protein. The role of the FeS cluster in Dna2 had been previously studied, where it was shown that loss of the FeS cluster eliminated Dna2’s nuclease activity and reduced ATPase activity. Considering the fragile nature of FeS clusters and the threat of oxidation, coupled with advancements in protein purification and FeS cluster biology, we asked whether the integrity of the FeS cluster was maintained during purification in the latter study. Therefore, using recombinant Dna2 WT and mutants purified using an optimized protocol, we characterized Dna2’s nuclease and helicase activities. Our in vitro data suggest that potent nuclease activity depends on the FeS cluster, but is not completely abolished in the FeS binding mutant, in contrast to previously reported data. Furthermore, the helicase activity is highly reduced upon loss of the FeS cluster, despite the helicase domain being distant from the FeS binding domain. In addition, we characterized the role of Dna2’s FeS cluster in Okazaki fragment maturation with nucleaseand replication-based assays. Our data suggests that the FeS cluster in Dna2 regulates its cleavage site in vitro, with WT Dna2 being able to cleave at the base of an Okazaki fragment, while the FeS binding mutant is unable to cleave at the base, which prevents the formation of a ligatable substrate and the completion of Okazaki fragment maturation. Taken together, our data suggest a critical role of the FeS cluster in Dna2 in regulating its cellular activities. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-152012 Dissertation Published Version Originally published at: Lutz, Richard Edward. Elucidating the role of the iron-sulfur cluster in the nuclease/helicase Dna2. 2018, University of Zurich, Faculty of Science.