Date of Award
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Agata Smogorzewska
Keywords
DNA replication, genome stability, cell cycle checkpoints, replication stress response
Abstract
The composition of the protein machinery that copies DNA, called the replisome, is dynamically regulated across S phase to promote the accurate and complete duplication of the genome before cell division. Work in our laboratory has uncovered one such dynamic process at replication forks experiencing replication stress, by which proteasome shuttle proteins DNA damage inducible 1 and DNA damage inducible 2 (DDI1/2) regulate the level of replication termination factor 2 (RTF2) at the replisome. When RTF2 is retained at stressed forks, the response to replication stress is compromised and replication forks fail to restart efficiently, leading to genome instability. However, the function of RTF2 within an active replisome has remained uncharacterized. Here, we examine the function of RTF2 across an unperturbed mammalian cell cycle, with a focus on RTF2’s activities at the replisome. We find that RTF2 is essential for maintenance of DNA replication elongation rates and control of replication origin firing. By comparing the composition of RTF2-deficient to RTF2-competent replisomes, we find that RTF2 recruits the heterotrimeric enzyme RNase H2 to the replication fork. Downstream of failed RNase H2 recruitment to the replisome, RTF2-deficient cells accumulate genomically embedded ribonucleotides. In DDI1/2-depleted cells, retention of RTF2 and RNase H2 at the replication fork leads to inefficient replication restart after fork stalling. We propose that retained RTF2-RNase H2 at the replication fork leads to inefficient RNA primer retention. Cells lacking RTF2 replicate DNA slowly and fire excess replication origins in S phase. We find that RTF2-deficient cells bypass the intrinsic S/G2 checkpoint and prematurely accumulate mitotic markers while replication proceeds. To understand the mechanism of checkpoint bypass in these cells, we performed an in silico protein interaction screen using AlphaFold-Multimer. We identified a high-confidence interaction between RTF2 and checkpoint kinase 1 (CHK1), an essential kinase that functions downstream of ataxia-telangiectasia and Rad3-related (ATR) in the intrinsic S/G2 checkpoint, which we confirmed in cells. Remarkably, cells lacking RTF2 fail to recruit CHK1 to replication forks. Abrogation of the putative RTF2 and CHK1 interaction leads to diminished, but not absent, recruitment of CHK1 to replication forks and increased accumulation of mitotic markers while replication proceeds. We find that RTF2 interacts with the essential CMG-interacting and CHK1-activating protein CLASPIN. Our findings show that CLASPIN, like RTF2, localizes CHK1 to replication forks and implicate CLASPIN in control of CHK1 activity during an unperturbed S phase. These findings uncover how RTF2 and CLASPIN poise CHK1 at replication forks to facilitate its switch-like activation by ATR, creating fork-localized CHK1 activity that fuels unperturbed S phase progression and promotes genome stability by maintaining the intrinsic S/G2 checkpoint. As ATR and CHK1 function to halt the cell cycle in response to replication stress, we next evaluated whether RTF2 may function in the replication stress response pathway. We find that RTF2-deficient cells fail to activate CHK1 in response to acute treatment with low doses of replication stress-inducing agents, suggesting that CHK1 localization to replication forks facilitates its dynamic activation by ATR in response to stress. We show that RTF2 and CHK1 are removed from replication forks experiencing low levels of replication stress and propose that this removal is necessary for robust, global activation of CHK1-mediated cell cycle arrest. We further identify a putative ATR phosphorylation site within RTF2’s amino acid sequence that is essential for maintaining DNA replication rates. Finally, we uncover a relationship between RTF2 and another checkpoint protein, monopolar spindle kinase 1 (MPS1), which monitors microtubule-kinetochore connection in the spindle association checkpoint (SAC). RTF2 and MPS1 share extensive co-dependency overlap in cancer cell lines and interact by immunoprecipitation. RTF2- deficient cells exhibit exquisite sensitivity to low-dose inhibition of MPS1, suggesting that RTF2 may further function in the SAC to prevent premature cell division.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Bronton, Cayla, "Functions of RTF2 in DNA Replication and Checkpoint Control" (2025). Student Theses and Dissertations. 836.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/836
Comments
Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy