On the train tracks of genomic DNA, double-strand breaks are like gaps on both rails. They are lethal. Survival mandates that double-strand breaks be repaired immediately.

One of the ways, therefore, of killing cancer cells is to stop the repair of double-strand breaks. Scientists are actively looking for ways to keep cancer cells with the BRCA mutations, in patients with breast and ovarian cancers, from engaging cellular repair mechanisms that seal double-strand breaks.

The defining catalyst for the double-strand break repair pathway is the large enzyme, polymerase theta (pol theta). New findings demonstrate a previously-unknown capability of pol theta that shows promise in developing treatments against hard-to-treat cancers. Because cancer cells rely on the pol theta pathway to survive and repair double-strand breaks, researchers have focused on pol theta, trying to find out how to inhibit this repair pathway.

Researchers at the University of Vermont (UVM), the University of Texas MD Anderson Cancer Center (MD Anderson), and Yale University have discovered that pol theta previously known to extend DNA in the repair of double-strand breaks, can also trim unpaired DNA in a direction-specific manner.

The findings are published in the article, “Human DNA polymerase theta harbors DNA end-trimming activity critical for DNA repair,” in the journal Molecular Cell.

Sylvie Doublié
Sylvie Doublié, PhD, professor of microbiology and molecular genetics at the UVM Larner College of Medicine and the UVM Cancer Center. [Doublié]
The double-strand break repair pathway called theta-mediated end joining (TMEJ) is catalyzed by pol theta. Pairing of two DNA strands over short homologous stretches generates unpaired ends in a direction commonly called 3’ (three prime). The nuclease responsible for cleaving the unpaired 3’ ends of DNA has been elusive so far. The current study reveals that this end-trimming function resides intrinsically within pol theta’s polymerase domain.

“Pol theta is a ‘hot’ enzyme right now,” said senior author and self-described “polymerase geek” Sylvie Doublié, PhD, professor of microbiology and molecular genetics at the UVM Larner College of Medicine and the UVM Cancer Center. “This is a new activity for pol theta; it’s an elegant way of solving the problem—you only need one enzyme.”

These findings could lead to the development of new therapeutic options, like the Poly-ADP-ribose polymerase (PARP) inhibitors—a class of drugs that have been used to treat breast and ovarian cancer.

“The cell has to decide which function needs to be applied and this trimming activity is a point of vulnerability for pol theta,” said Doublié. Doublié’s team is focused on figuring out conditions where one reaction of pol theta can be encouraged over the other.

A potential role for such an inhibitor would be to improve ionizing radiation therapy in cancer patients with BRCA1 or BRCA2 mutations.

Pol theta
Three-dimensional structure of the pol theta enzyme. [Brian Eckenroth, Doublie Lab, University of Vermont]
Karl Zahn, PhD, a former doctoral student in Doublié’s lab and now a postdoctoral fellow at Yale, based upon initial data that hinted at the dual function of pol theta several years ago, engaged the expertise of Richard Wood, PhD, professor of epigenetics and molecular carcinogenesis at MD Anderson. Wood and Doublié have a long-term collaboration, funded by a Program Project grant from the National Cancer Institute.

“It was an unexpected finding, and the biochemistry makes sense, suggesting a way to inhibit the DNA repair process orchestrated by pol theta,” said Wood.

The end-trimming activity allows pol theta to process DNA ends prior to DNA synthesis. The activity depends on metal ions, deoxynucleotides, and pol active-site residues and is not simply a reverse of DNA polymerization (pyrophosphorolysis) or proofreading activity often associated with DNA polymerases.

“We reveal a nimble strategy of substrate processing that allows pol theta to trim or extend DNA depending on the DNA repair context,” the authors noted.

“The trimming reaction is rapid, and many people missed it,” said Doublié, adding that the research team’s patience and work paid off. “‘Chance favors only the prepared mind,'” she said, quoting the late French scientist Louis Pasteur.

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