Date of Award
Doctor of Philosophy (PhD)
Faithful DNA replication and repair are essential for maintaining genome stability and preventing various diseases including cancer. Both processes are executed by numerous redundant mechanisms to ensure that these processes are uninterrupted even when a primary mechanism fails. Despite this, they are not immune to challenges and failures leading to DNA damage and genome instability. These problems are more evident at the difficult-to-replicate regions of the genome such as the telomeres that cap and protect linear chromosome ends. Additionally, topological structures such as RNA:DNA hybrids, commonly referred to as R loops, can also present severe challenges to the DNA replication and repair machineries. Herein we report the functions of two distinct DNA replication and repair proteins—flap endonuclease 1 (FEN1) and ribonuclease H1 (RNH1)—that preserve genome stability. First, we show that FEN1 limits telomere fragility in leading strand replicated telomeres. This is mediated by its flap endonuclease activity independent of its gap endonuclease activity and C-terminal interactions. We show that the fragility phenotype is increased by RNA polymerase II inhibition and rescued by ectopic RNH1 expression. Because the telomere is transcribed and can form hybrids, these data suggest that the FEN1-mediated telomere fragility depends on RNA:DNA hybrids that accumulate from co-directional replisome-RNAP collision at the leading strand replicated telomere. These findings are the first to assign a leading strand specific function of FEN1, which is a canonical lagging strand protein. Second, we uncover a novel role for human RNH1 in DNA replication in the nucleus. We show that RNH1 depletion results in a global DNA damage response as well as telomere loss phenotype. Because RNH1 resolves RNA:DNA hybrids, we measured those hybrid levels and found that they increase upon RNH1 depletion. Given these hybrids could pose barriers to a moving replication machinery, we interrogated replication efficiency and discovered that RNH1 facilitates the replication fork movement, possibly by clearing hybrids. These data shed light onto the role of RNH1 in global DNA replication. Together, our work underscore the complexity of DNA replication and repair processes and highlight the varied roles that FEN1 and RNH1 play to maintain genome stability.
Chair and Committee
Sheila A. Stewart
Peter M. Burgers, Nima Mosammaparast, Alessandro Vindigni, Zhongsheng You,
Parajuli, Shankar, "DNA Replication Challenges: Telomeres and R loops" (2017). Arts & Sciences Electronic Theses and Dissertations. 1209.