Biology and Biomedical Sciences: Molecular Cell Biology
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Peter M Burgers
The work outlined in this dissertation focuses on two distinct areas that are important for genome stability. Both areas focus on DNA repair pathways that require the action of nucleases, specifically Exonuclease 5 and Ribonuclease H2. First, I describe the biochemical and molecular characterization of the novel Exonuclease 5 family of enzymes from S. cerevisiae, S. pombe, and humans. The Exo5 family consists of bi-directional single-strand DNA specific exonucleases that all contain an iron-sulfur cluster as a structural motif and all have various roles in DNA metabolism. In the Saccharomycetales order that includes the budding yeast, S. cerevisiae, Exo5 is a mitochondrial protein that is essential for mitochondrial genome maintenance. In an unrelated yeast species, Schizosaccharomyes pombe, Exo5 is important for both nuclear and mitochondrial DNA metabolism. The human ortholog is important for nuclear genome stability, and for DNA repair. The work outlined in Chapter II of this Dissertation establishes Exo5 as a protein that is important for DNA metabolism.
The second area of study outlined in Chapters III and IV is related to the phenomenon of ribonucleotide incorporation into the genome by replicative polymerases, and these chapters focus on the enzymes that remove these noncanonical nucleotides. Ribonucleotides are incorporated into DNA by the replicative DNA polymerases at frequencies of about 2 per kb, which makes them by far the most abundant form of potential DNA damage in the cell. Their removal is essential for restoring a stable intact chromosome. In Chapter III, I present a complete biochemical reconstitution of the ribonucleotide excision repair (RER) pathway with enzymes purified from Saccharomyces cerevisiae. I highlight the requirement for RNase H2 in the process of RER and investigate the redundancies at different steps of repair. Also outlined in this dissertation is the dissection of the different functions of RNase H2 in RER and in the removal of RNA-loops in DNA, and implications for genome instability in human diseases that are affected for these activities.
Chapter IV of this dissertation discusses work on an alternative pathway for ribonucleotide removal from the genome by Topoisomerase I. In S. cerevisiae, deletion of rnh201, the catalytic subunit of RNase H2, results in the persistence of ribonucleotides remain in the genome, which leads to ~100-fold increase in the frequency of 2-5 bp deletions at di-nucleotide repeat sequences. These deletions are dependent on topoisomerase I (Top1) activity. Here we present an in vitro reconstitution of the mechanism of Top1-dependent deletions at di-nucleotide repeat sequences and a mechanism for Top1-initiated removal of ribonucleotides outside of the context of these repeat sequences in S. cerevisiae. Top1 attack at a ribonucleotide leads to the formation of a 2', 3' cyclic phosphate terminated ssDNA nick, followed by subsequent formation of a Top1-cleavage complex (Top1-cc) upstream of the 2', 3' cyclic phosphate. If the ribonucleotide is in the context of a di-nucleotide repeat, there can be realignment of the DNA allowing for religation and release of Top1, leading to a 2-nucleotide deletion. If the ribonucleotide resides outside a repeat sequence, the realignment is not possible and a different pathway must repair the Top1-cc. Tdp1-dependent repair of Top1-cc requires prior proteolytic processing of the Top1-cc before it can be removed leaving a 3'-phosphate that can be removed by Tpp1, Apn1, or Apn2 forming a substrate suitable for repair by DNA polymerase δ, FEN1 and DNA ligase.
Sparks, Justin Lee, "Functional Roles of Nucleases in DNA Metabolism and Genome Stability" (2014). All Theses and Dissertations (ETDs). 1349.