Date of Award
Doctor of Philosophy (PhD)
DNA replication must occur efficiently and timely every cell cycle to protect the integrity of the genome. Stalled or slowed replication forks lead to replication stress that can cause replication fork collapse, and potentially genome instability. Scattered throughout the genome are tightly bound proteins, such as transcription factors, that are necessary for cell function and survival. These proteins have the potential to impede timely DNA replication. Furthermore, genomic DNA is packaged around histone octamers into structures called nucleosomes that both compact the DNA and provide an additional layer of information and regulation termed epigenetics. Thus, DNA replication is not only tasked with maintaining the integrity of the DNA sequence but also preserving and duplicating the epigenetic information encoded by nucleosomes. This task is made all the more difficult as nucleosomes are displaced from in front of the replication fork and reassembled behind the fork. Although this tightly regulated choreography removes nucleosomes from the path of the leading strand, nucleosomes are redeposited directly in the path of the lagging strand DNA polymerase _ (Pol _), generating a potential obstacle to overcome. Also, because of the reverse directionality of lagging strand replication, the lagging strand must replicate DNA discontinuously, resulting in long stretches of single stranded DNA (ssDNA) that can fold into stable DNA secondary structures, such as hairpins or G-quadruplexes, that are known barriers to DNA synthesis.
Successful DNA replication requires carefully regulated mechanisms to overcome these numerous obstacles that naturally occur throughout chromosomal DNA. Herein, I describe work detailing how Pif1, a 5� to 3� DNA helicase, is able to remove a large variety of barriers to Pol _ DNA synthesis: DNA hairpins, G-quadruplexes, nucleosomes, and the general transcription factors, Reb1 and Tbf1. The activity of Pif1 is necessary for efficient DNA replication at all of these barriers except DNA hairpins and Tbf1, for which the DNA binding activity of the ssDNA binding protein RPA was sufficient. However, my work also highlighted two potential problems with Pif1 stimulated DNA replication. First, while unwinding DNA secondary structures, the 5� to 3� nature of Pif1 DNA helicase activity results in a Pif1-Pol _ head-on conflict that stimulates the exonuclease activity of the polymerase and results in the degradation of newly synthesized DNA. I show that RPA is required to prevent this conflict by a currently unknown mechanism. Second, Pif1 disruption of nucleosomes could lead to re-replication of newly synthetized DNA, a futile activity that needs to be suppressed to prevent epigenetic loss. However, the presence of the endonuclease FEN1 was able to prevent Pif1 stimulation of DNA synthesis through a nucleosome. FEN1 achieves this inhibition by traveling along with Pol _ through binding to the polymerase processivity factor PCNA and cleaving the short 5� tails generated during strand displacement, eliminating the entry point for Pif1. This activity would protect the cell from potential epigenetic loss caused by Pif1 interference during lagging strand replication. Combined, this work highlights a potential mechanism to overcome multiple natural barriers during DNA replication using the DNA helicase Pif1.
Chair and Committee
Roberto Galletto Peter Burgers
Timothy Lohman, Nima Mosammaparast, Alessandro Vindigni,
Sparks, Melanie Anne, "Bumpy Road Ahead: Overcoming DNA Replication Obstacles One Barrier at a Time" (2020). Arts & Sciences Electronic Theses and Dissertations. 2348.