The objective of this proposal is to understand the mechanisms that govern DNA replication fork reversal and restart following genotoxic stress induction. Fork reversal and restart is an emerging model to explain how stalled or damaged replication forks are processed upon replication inhibition. DNA replication inhibitors are widely used for chemotherapy, but they are also highly toxic to normal cells. Understanding the mechanisms that induce and carry out fork reversal and restart is thus critical for identifying novel molecular targets to sensitize caner cells to lower drug dosages that are not toxic to normal cells. Drugs that inhibit DNA topoisomerase 1 (TOP1) are currently used to treat cancer. Recent studies revealed that TOP1 inhibitor cytotoxicity is modulated by replication fork reversal. We discovered that the human RECQ1 helicase promotes the restart of reversed forks after TOP1 inhibition, and that the poly(ADPribosyl)ation activity of PARP1 stabilizes forks in their regressed state by limiting their restart by RECQ1. Within this application we show that regression of replication forks is not limited to TOP1 inhibition. Indeed, both hydroxyurea (HU) and mitomycin C (MMC) treatment induce replication fork regression. These findings suggest that fork reversal and restart represents a general response to different classes of cancer chemotherapeutics. Thus, elucidating the mechanisms that govern fork regression and restart will have broad impact in our understanding of several chemotherapeutic modalities. Our initial studies also revealed that an additional mechanism relying on the DNA processing activity of the Dna2 nuclease/helicase is implicated in the process when cells are challenged with HU. Therefore, we hypothesize that at least two alternative mechanisms control the restart of reversed forks following replication stress induction. We will test these ideas using a unique combination of single-molecule DNA fiber, electron microscopy, and biochemical approaches:
Aim 1 will determine whether fork reversal and restart in response to HU and MMC treatment occurs via the same central PARP1- and RECQ1-dependent mechanism seen in response to TOP1 inhibition.
Aim 2 will determine how reversed fork processing mediates fork restart. We will focus on the human Dna2 and Mre11 nucleases since their involvement in this process is strongly supported by our preliminary findings and available literature.
Aim 3 will elucidate the mechanism of replication fork reversal. We hypothesize that fork reversal is actively driven by selected factors. We will determine the requirement for Fanconi anemia protein M (FANCM), HepA-related protein (HARP, alias SMARCAL1), as well as the human BLM and WRN helicases, in this process. These helicases are strong candidates because they promote replication fork reversal in vitro, are implicated in replication stress response, and are linked to well-characterized genome-instability disorders, but their contribution in fork reversal and restart remains to be determined. Completion of these aims will shed new light on the detailed mechanisms by which replication responds to genotoxic stress.

Public Health Relevance

This project will address fundamental questions about the molecular mechanisms of replication stress response to an important subset of chemotherapeutic agents that act by inhibiting DNA replication. This knowledge is crucial for the design of novel molecularly guided treatments that will improve current chemotherapeutic regimens based on the use of DNA replication inhibitors.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Willis, Kristine Amalee
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Saint Louis University
Schools of Medicine
Saint Louis
United States
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Kreienkamp, Ray; Graziano, Simona; Coll-Bonfill, Nuria et al. (2018) A Cell-Intrinsic Interferon-like Response Links Replication Stress to Cellular Aging Caused by Progerin. Cell Rep 22:2006-2015
Quinet, Annabel; Carvajal-Maldonado, Denisse; Lemacon, Delphine et al. (2017) DNA Fiber Analysis: Mind the Gap! Methods Enzymol 591:55-82
Vindigni, Alessandro; Lopes, Massimo (2017) Combining electron microscopy with single molecule DNA fiber approaches to study DNA replication dynamics. Biophys Chem 225:3-9
Elango, Rajula; Sheng, Ziwei; Jackson, Jessica et al. (2017) Break-induced replication promotes formation of lethal joint molecules dissolved by Srs2. Nat Commun 8:1790
Brickner, Joshua R; Soll, Jennifer M; Lombardi, Patrick M et al. (2017) A ubiquitin-dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair. Nature 551:389-393
Pasero, Philippe; Vindigni, Alessandro (2017) Nucleases Acting at Stalled Forks: How to Reboot the Replication Program with a Few Shortcuts. Annu Rev Genet 51:477-499
Quinet, Annabel; Lemaçon, Delphine; Vindigni, Alessandro (2017) Replication Fork Reversal: Players and Guardians. Mol Cell 68:830-833
Lemaçon, Delphine; Jackson, Jessica; Quinet, Annabel et al. (2017) MRE11 and EXO1 nucleases degrade reversed forks and elicit MUS81-dependent fork rescue in BRCA2-deficient cells. Nat Commun 8:860
Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna et al. (2017) Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork. J Biol Chem 292:15216-15224
Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa et al. (2016) Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors. Elife 5:

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