Imaging tool reveals novel insights into DNA replication stress response

MD Anderson Research News April 27, 2026

• Reversed DNA replication forks protect against stress to maintain genomic stability and serve as central components to cancer resistance, disease suppression, aging and immunotherapy response
• Researchers developed the RF-SIRF imaging tool to map reversed forks with single-cell resolution and within their native cell environment
• RF-SIRF highlighted unique epigenetic signals that provide insights into how cells respond to replication stress
• Understanding DNA stress response advances precision medicine for more targeted therapies 

Researchers at The University of Texas MD Anderson Cancer Center have developed a new imaging method, known as RF-SIRF, that quantitatively detects and maps reversed DNA replication forks with single-cell resolution. The results also demonstrated a unique epigenetic code for DNA replication stress that can be further examined to understand mechanisms of genomic stability, aging and treatment response.

The study, published in Nature Communications, was led by Katharina Schlacher, Ph.D., associate professor of Cancer Biology.

“By capturing reversed DNA replication forks in their spatiotemporal context, our new assay identifies site-specific epigenetic signatures,” Schlacher said. “This technology provides a unique lens, enabling scientists to decode cancer-specific DNA replication stress dynamics and their crosstalk with inflammation and transcription programs, representing a major step in precision oncology.”

What are reversed DNA replication forks and why are they important?

DNA replication happens at replication forks, where the double helix is unzipped and new strands are built. However, these forks can sometimes collapse because of certain stressors, like DNA damage, aging, disease or cancer treatments.

To protect against genomic instability from replication stress, cells can reverse the replication forks, creating a four-way structure that temporarily stalls the process to promote damage tolerance and avoid the formation of DNA double strand breaks.

While reversed forks can be helpful in many cases, they also can be harmful in individuals with certain genetic backgrounds — including those with BRCA1/2 fork protection-defective cells — because their formation controls cell sensitivity and response to treatments such as chemotherapy and immunotherapy.

Even though reversed forks have been studied in vitro and are known to play key roles in human health and disease, there currently are no robust, high-resolution tools that can examine their molecular mechanisms or dynamics within native cells. This led Schlacher and colleagues to develop RF-SIRF, a quantitative method to harness the unique four-way structure of the reversed DNA forks in order to map them with single-cell resolution.

What are the implications of the RF-SIRF imaging tool?

RF-SIRF allows researchers to study reversed forks in their native cell environment, taking into consideration their location, timing, geometric structure and protein interactions. Furthermore, the researchers demonstrated that reversed forks have a distinct, stress-specific epigenetic codeof signals that are different from those used in normal gene transcription.

These specific epigenetic signals recruit DNA stress response proteins to stalled replication forks, highlighting a new way to understand the mechanisms and dynamics of how cells prioritize these responses and develop ways to identify potential cancer-specific therapeutic targets.

“Targeting cancer therapy resistance remains one of the holy grails in cancer therapy,” Schlacher said. “For years, we’ve known that reversed forks dictate cancer therapy outcomes in BRCA-mutant cancer cells. We now have a method to directly study these enigmatic DNA structures with single-cell resolution, allowing us to comprehensively visualize hidden resistance and inflammation mechanisms and to directly test therapies that will overcome resistance at the molecular level.”

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The study was funded by the National Institute of Environmental Health Sciences (NIEHS) and the Cancer Prevention and Research Institute of Texas (CPRIT). A full list of collaborating authors and their disclosures can be found with the paper in Nature Communications.