Related Experiment Video
Updated: May 5, 2026
Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging
Published on: April 28, 2021
Tumor cell death mediated by peptides that recognize branched intermediates of DNA replication and repair
Mamon Dey1, Sukanya Patra, Leo Y Su
1Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, California, United States of America.
Abstract:
Effective treatments for cancer are still needed, both for cancers that do not respond well to current therapeutics and for cancers that become resistant to available treatments. Herein we investigated the effect of a structure-selective d-amino acid peptide wrwycr that binds replication fork mimics and Holliday Junction (HJs) intermediates of homologous recombination (HR) in vitro, and inhibits their resolution by HJ-processing enzymes. We predicted that treating cells with HJ-binding compounds would lead to accumulation of DNA damage. As cells repair endogenous or exogenous DNA damage, collapsed replication forks and HJ intermediates will accumulate and serve as targets for the HJ-binding peptides. Inhibiting junction resolution will lead to further accumulation of DNA breaks, eventually resulting in amplification of the damage and causing cell death. Both peptide wrwycr and the related wrwyrggrywrw entered cancer cells and reduced cell survival in a dose- and time-dependent manner. Early markers for DNA damage, γH2AX foci and 53BP1 foci, increased with dose and/or time exposure to the peptides. DNA breaks persisted at least 48 h, and both checkpoint proteins Chk1 and Chk2 were activated. The passage of the cells from S to G2/M was blocked even after 72 h. Apoptosis, however, was not induced in either HeLa or PC3 cells. Based on colony-forming assays, about 35% peptide-induced cytotoxicity was irreversible. Finally, sublethal doses of peptide wrwycr (50-100 µM) in conjunction with sublethal doses of several DNA damaging agents (etoposide, doxorubicin, and HU) reduced cell survival at least additively and sometimes synergistically. Taken together, the results suggest that the peptides merit further investigation as proof-of-principle molecules for a new class of anti-cancer therapeutics, in particular in combination with other DNA damaging therapies.
Insights
New peptide compounds targeting DNA repair mechanisms show promise as novel cancer treatments. These peptides induce DNA damage and cell death, particularly when combined with existing therapies, offering a new avenue for drug-resistant cancers.
Area of Science:
- Molecular Biology
- Cancer Research
- Drug Discovery
Background:
- Effective cancer treatments are needed for drug-resistant and non-responsive cancers.
- Homologous recombination (HR) is a critical DNA repair pathway.
- Holliday Junctions (HJs) are key intermediates in HR that can be targeted for therapeutic intervention.
Purpose of the Study:
- To investigate the anti-cancer effects of a structure-selective peptide (wrwycr) that binds to replication fork mimics and Holliday Junctions (HJs).
- To determine if targeting HJ resolution can lead to DNA damage accumulation and cancer cell death.
- To evaluate the peptide's efficacy alone and in combination with DNA damaging agents.
Main Methods:
- In vitro binding assays with replication fork mimics and HJs.
- Treatment of cancer cells (HeLa, PC3) with peptides wrwycr and wrwyrggrywrw.
- Assessment of DNA damage markers (γH2AX, 53BP1 foci, DNA breaks).
- Cell cycle analysis (S to G2/M progression).
- Colony-forming assays to determine cytotoxicity.
- Combination studies with DNA damaging agents (etoposide, doxorubicin, HU).
Main Results:
- Peptides wrwycr and wrwyrggrywrw entered cancer cells and reduced cell survival in a dose- and time-dependent manner.
- Increased DNA damage markers (γH2AX, 53BP1 foci) and persistent DNA breaks were observed.
- Cell cycle progression from S to G2/M was blocked, and Chk1/Chk2 were activated.
- Apoptosis was not induced, but approximately 35% irreversible cytotoxicity was measured.
- Sublethal doses of peptide wrwycr synergized with DNA damaging agents to reduce cell survival.
Conclusions:
- The peptide wrwycr acts as a proof-of-principle molecule for a novel class of anti-cancer therapeutics.
- Targeting Holliday Junction resolution with peptides can induce significant DNA damage and cytotoxicity in cancer cells.
- These peptides show potential for combination therapy with existing DNA damaging agents to overcome treatment resistance.
Related Concept Videos
02:36DNA Damage Can Stall the Cell Cycle
02:36DNA Damage can Stall the Cell Cycle
01:17The Extrinsic Apoptotic Pathway
01:31The Intrinsic Apoptotic Pathway
02:25Overview of DNA Repair
Chemically...
01:30Overview of Cell Death
Cell death was observed in the early 19th century, but there was no experimental evidence to prove it. In 1842, Carl Vogt first discovered cell death in a metamorphic toad; however, it was not termed ‘cell death.’ Scientists discovered different cell death pathways only in the...