# Okazaki fragment maturation: mutagenesis and cell survival

> **NIH NIH R01** · BECKMAN RESEARCH INSTITUTE/CITY OF HOPE · 2023 · $547,800

## Abstract

ABSTRACT
The long-term goal of this project is to define the molecular mechanisms of an error-prone, stress-induced
Okazaki fragment maturation (OFM) pathway by which cancer cells counteract replication stress and survive.
Replication stress is a hallmark of cancer cells and has been considered the Achilles' heel for cancer treatment
such as radio- and chemotherapy. Under elevated temperature stress, yeast cells mutant for flap endonuclease 1
(FEN1 in humans or RAD27 in yeast) activate DNA damage response pathways to block cell proliferation and
induce cell senescence and death; however, a subpopulation of cells can overcome these barriers and escape
otherwise lethal conditions. Genome-wide mutations and rearrangements have been suggested as a major
molecular mechanism that drives this evolution. However, how such spontaneous mutations are acquired in cells
under replication stress is a long-standing question. Recently, we identified an error-prone, 3' flap OFM pathway
that is activated in response to stress to support cell survival and fuel cellular evolution; its induction leads to
genome-wide mutagenesis and suppression of restrictive growth temperature-induced lethality, a process
mimicking that of cancer cells acquiring drug resistance. This led us to a model in which OFM can go in two
ways, which may dictate the fate of cells, including human cancer cells: a 5' flap-based, error-free process or an
alternative 3' flap-based, stress-induced, and error-prone process. However, key components that drive such
flap dynamics remain undefined. The objectives of the proposed project are to define the key enzymes that
catalyze 3' flap formation and cleavage in mammalian cells and to provide proof of concept that suppressing
alternative 3' flap OFM can prevent drug resistance in human cancer cells. Further preliminary data gathered to
support this grant application show that 3' flap OFM is conserved in both yeast and human cells. We observed
that anti-cancer EGFR tyrosine kinase inhibitors activated the ATM/CHK2 DNA damage checkpoints in human
lung cancer cells. Using yeast genetic screening, we identified Pif1 (PIF1 in humans) and Sgs1 (BLM and WRN
in humans) as helicases for 5' to 3' flap transformation and Rad1 (XPF in humans) and Mus81 (MUS81 in
humans) as 3' nucleases for 3' flap cleavage, in addition to the 3' nuclease activity of Pol . Therefore, our central
hypothesis is that unprocessed 5' flaps in mammalian cells activate ATM/ATR and CHK1/2 signaling to recruit
and stimulate PIF1, BLM, and WRN and/or other helicases for transforming 5' flaps into 3' flaps for nucleolytic
degradation by 3' nucleases including Pol , XPF, and/or MUS81, and that blocking the 3' flap OFM pathway
will suppress DNA mutations and thus prevent drug resistance. To test this, we will: i) determine the roles of
helicases PIF1, BLM, and WRN in 3' flap formation and induction of alternative OFM; ii) define the functional
distribution of 3' nucleases Pol , XPF, and MUS81...

## Key facts

- **NIH application ID:** 10636417
- **Project number:** 1R01CA279840-01
- **Recipient organization:** BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
- **Principal Investigator:** BINGHUI SHEN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $547,800
- **Award type:** 1
- **Project period:** 2023-04-01 → 2028-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10636417

## Citation

> US National Institutes of Health, RePORTER application 10636417, Okazaki fragment maturation: mutagenesis and cell survival (1R01CA279840-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10636417. Licensed CC0.

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