A new experimental assay can help scientists find the precise locations of repair of DNA damage caused by radiation and common chemotherapies. The invention could lead to better cancer drugs or improvements in the potency of existing ones, and also to repair methods for radiation damage to DNA.

When the common chemotherapy drugs cisplatin or oxaliplatin hit cancer cells, they damage DNA so that the cells can’t replicate. But cancer cells have ways to repair the DNA, reducing the effectiveness of the cancer drugs.

Now researchers at the University of North Carolina (UNC) School of Medicine and UNC Lineberger Comprehensive Cancer Center have developed a method for finding where this DNA repair happens throughout all of human DNA.

The findings, published in the journal Genes & Development, offers scientists a potential way to find and target the proteins that cancer cells use to work around therapeutic drugs. The new method could lead to more effective and better tolerated classes of cancer therapeutics.

The research, led by Aziz Sancar, MD, PhD, the Sarah Graham Kenan Professor of Biochemistry and Biophysics, marks the first time scientists have been able to map the repair of DNA damage over the entire human genome.

“Now we can say to a fellow scientist, ‘tell us the gene you’re interested in or any spot on the genome, and we’ll tell you how it is repaired,’” said Sancar, co-senior author and member of the UNC Lineberger Comprehensive Cancer Center.

Mapping the repair of DNA damage

When DNA is damaged, cells use many enzymes to cut the strand of DNA and excise the damaged fragment. Then, other enzymes repair the original DNA so that the cells can function properly. The researchers on Sancar’s team decoded this process in a series of experiments.

1. They used purified enzymes to discover how this process happens in DNA damaged by UV irradiation and by chemotherapeutic drugs such as cisplatin and oxaliplatin.
2. They found that a particular protein called TFIIH bound tightly to the excised (cut out) damaged DNA fragment in the test tube.
3. Through a series of sophisticated experiments with human skin cells, they exposed the cells to ultraviolet radiation and used an antibody against the enzyme TFIIH to isolate the enzyme complex with the excised DNA damage. Then he created experimental techniques to pull the enzyme, as well as the excised DNA fragment it was bound to, from the cells.
4. They then sequenced this fragment.
5. Using computational biology, they analyzed where the DNA repair happened throughout the entire genome — thus generating a human genome repair map for the first time.

Because UV radiation and common chemotherapy drugs such as cisplatin cause DNA damage in similar ways, Sancar’s team is now using their new DNA excision repair method — called XR-Seq — to study cells affected by cisplatin. They also hope to use it to study the biochemical reactions in animal models with the goal of finding the specific mechanisms that allow cancer cells to repair DNA damage to survive, and then to use these methods to make cancer cells more sensitive to existing drugs to help patients.

The role of noncoding DNA  in repair

The research also revealed that “noncoding DNA” portions of the genome, previously thought to do very little, are actually part of this repair process.

On chromosomes, DNA forms genes that create proteins — the building blocks of life. Between these genes, there are DNA sequences that were considered useless. The researchers found that proteins actually bind to these other DNA sequences, and this affects other nearby or far-away DNA regulatory sequences, which are also being repaired. If they’re being repaired, they’re likely important, so the researchers now have a method  finding their locations throughout the genome.

The National Institutes of Health funded this research.

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Abstract of Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution

We developed a method for genome-wide mapping of DNA excision repair named XR-seq (excision repair sequencing). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine–pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled excision repair or general excision repair isolated the contribution of each pathway to the overall repair pattern and showed that transcription-coupled repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA repair in human cells.