# Role of DNA Replication Stress in Genome Instability and Cancer

> **NIH NIH R01** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2020 · $284,489

## Abstract

PROJECT SUMMARY
During the development of cancer, the incipient tumors exhibit signs of replication stress, including
deregulation of origin firing, stalled forks, DSBs, and activation of p53. To what extent replication stress is a
driving force in cancer can only be understood by further characterization of the pathways that stabilize and
repair replication forks, allow restart for completion of replication, and induce a protective senescence or
apoptotic state in precancerous cells. While HR (homologous recombination) proteins are involved in repair of
stressed replication forks, their precise roles and regulation at forks are poorly understood compared to their
function at DSBs. We will address these issues using the FA/BRCA (Fanconi anemia/Breast cancer) pathway
of replication fork rescue as a focus. Recently, we demonstrated that DNA2 helicase/nuclease, plays a role in
the FA/BRCA pathway. DNA2 is involved along with the MRN complex and BLM or WRN in resection of
DSBs, giving rise to the 3’ overhangs that induce the DNA damage stress response. These overhangs also
recruit Rad51 to form filaments that participate in strand invasion during recombination and filament formation
during stressed replication fork protection. Since DNA2 is also essential for DNA replication, we hypothesize
that an important part of its HR/resection-related function is at genome destabilizing ssDNAs, gaps, DSBs or
reversed forks arising during replication stress. Depletion of DNA2 suppresses chemosensitivity in FANCD2-
deficient cells, and we will study the basis of this antagonism, which we propose is related to regulation of
DNA2 resection by FA/BRCA pathway components. In Aim 1, we will use cytogenetics, genomics, and
biochemistry to study the interplay of FANCD2 and DNA2 in choice of pathways (HR, NHEJ, and alt-NHEJ)
that may compete for DNA ends or gaps arising during FA/BRCA repair of stalled replication forks. In Aim 2,
we will further define the DNA2/FANCD2 interplay in replication fork protection and restart using iPOND and
single DNA molecule analysis, highlighting novel analysis of FRA16D, a common fragile site that is unstable
in FANCD2 deficient cells. In Aim 3, we describe an innovative, high resolution approach to studying the
contribution of these factors to replication dynamics based on stalling replication at a chromosomal, site-
specific protein/DNA replication block. A novel tool for our studies will be the DNA2 inhibitor we developed,
which may also have therapeutic potential by sensitizing certain tumors to chemotherapies that cause
replication stress.

## Key facts

- **NIH application ID:** 9954111
- **Project number:** 5R01GM123554-04
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Judith L CAMPBELL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $284,489
- **Award type:** 5
- **Project period:** 2017-07-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9954111

## Citation

> US National Institutes of Health, RePORTER application 9954111, Role of DNA Replication Stress in Genome Instability and Cancer (5R01GM123554-04). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9954111. Licensed CC0.

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