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Updated: Dec 17, 2025
Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay
Published on: February 3, 2022
Inhibition of the translesion synthesis polymerase REV1 exploits replication gaps as a cancer vulnerability
Sumeet Nayak1, Jennifer A Calvo1, Ke Cong1
1Molecular Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Abstract:
The replication stress response, which serves as an anticancer barrier, is activated not only by DNA damage and replication obstacles but also oncogenes, thus obscuring how cancer evolves. Here, we identify that oncogene expression, similar to other replication stress-inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness. DNA fiber analysis and electron microscopy reveal that activation of translesion synthesis (TLS) polymerases restricts replication fork slowing, reversal, and fork degradation without inducing replication gaps despite the continuation of replication during stress. Consistent with gap suppression (GS) being fundamental to cancer, we demonstrate that a small-molecule inhibitor targeting the TLS factor REV1 not only disrupts DNA replication and cancer cell fitness but also synergizes with gap-inducing therapies such as inhibitors of ATR or Wee1. Our work illuminates that GS during replication is critical for cancer cell fitness and therefore a targetable vulnerability.
Insights
Oncogenes create DNA gaps, harming cancer cell fitness. Activating translesion synthesis (TLS) polymerases prevents these gaps, a key cancer vulnerability. Targeting TLS factors like REV1 offers a new cancer therapy strategy.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- The replication stress response is a crucial anticancer mechanism activated by DNA damage, replication obstacles, and oncogenes.
- Oncogene activation can obscure cancer evolution by inducing replication stress.
- Understanding how cancer cells cope with oncogene-induced replication stress is vital for developing effective therapies.
Purpose of the Study:
- To investigate the role of oncogene expression in inducing replication stress and its impact on cancer cell fitness.
- To elucidate the mechanism by which cancer cells manage single-stranded DNA (ssDNA) gaps during oncogene-induced replication stress.
- To evaluate the therapeutic potential of targeting the translesion synthesis (TLS) pathway for cancer treatment.
Main Methods:
- DNA fiber analysis and electron microscopy were used to study replication fork dynamics and ssDNA gap formation.
- Investigated the role of translesion synthesis (TLS) polymerases in managing replication stress.
- Utilized a small-molecule inhibitor targeting the TLS factor REV1 in cancer cell models.
- Assessed the synergistic effects of REV1 inhibition with ATR or Wee1 inhibitors.
Main Results:
- Oncogene expression induces single-stranded DNA (ssDNA) gaps, reducing cancer cell fitness.
- Activation of TLS polymerases suppresses replication fork slowing, reversal, and degradation, preventing gap formation during stress.
- Inhibiting the TLS factor REV1 disrupts DNA replication and impairs cancer cell fitness.
- REV1 inhibition synergizes with therapies targeting ATR or Wee1, which induce DNA gaps.
Conclusions:
- Gap suppression (GS) during replication is fundamental for cancer cell fitness.
- Oncogene-induced ssDNA gaps represent a targetable vulnerability in cancer.
- Targeting TLS factors like REV1, in combination with gap-inducing therapies, offers a promising strategy for cancer treatment.
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