Fluorescent marker highlights potency of DNA breaks

By Ruth SoRelle, M.P.H.

Using a new technique that makes cells with DNA damage appear green, Baylor College of Medicine researchers found that breaks of double-stranded DNA occur much less frequently than previously estimated but are much more potent in causing cells to go down the wrong path, as when they become malignant. A report on this work appeared recently in the journal Nature Genetics.

Susan Rosenberg, Ph.D., professor of molecular and human genetics at BCM, and Jeanine Pennington, Ph.D., a postdoctoral fellow at BCM, developed the technology in Escherichia coli (E. coli) by attaching a gene for green fluorescent protein to a gene that promotes the SOS DNA damage response. The SOS response is generated when the cell detects breaks in the double-strands of DNA. SOS is designed to repair these breaks and let the genome reproduce normally in new cells. The fluorescent protein caused cells to look green when SOS was activated.

Breaks occur less than thought

Using the green as a marker, Rosenberg and Pennington were able to identify cells in which such breaks occurred naturally. Previously, scientists estimated that such breaks occurred as often as once each time the genome was replicated in E. coli and as many as 50 times each time the much longer and more complicated human genome was replicated.

Using this technique, which identifies approximately 27 percent of cells in which such damage occurs, the scientists estimated that 1 percent of cells get one or more double-stranded breaks in their DNA with each genome replication, Rosenberg said. That is 20- to 100-fold less than previous estimates, she said.

The technique is important because it allows scientists to actually identify the DNA damage as it occurs in its natural state – not when they have insulted the cells with damaging chemicals, said Rosenberg. While she has demonstrated that such damage occurs naturally in E. coli, she said it needs to be tested in human cells as well.

"Most people believe that the majority of genome instability comes from normal biological processes that you cannot avoid, such as replicating DNA," she said. "This allows us to see what the cell does to itself when it's in a relatively normal environment."

The danger behind double-stranded breaks

It also allows them to quantify what is happening from direct observation. "What this really means is that we know how much genome rearrangement happens per replication," she said. "And we also know that most of it comes from double-stranded breaks. Every double-stranded break is 100 times more dangerous than we realized it was before."

For example, some tumors, such as breast cancer, can be traced to a breakdown in the repair of double-stranded breaks in DNA. This could change how one looks at such issues, she said.

Unexpected findings

She also used the technique to study what happened to cells that had undergone DNA repair after a double-stranded break. Thirty percent of them grew and could replicate.

"What we didn't expect was that the other 70 percent were not dead. They could not proliferate, but by all measurements, they were still alive – even after eight hours in a culture medium (eight generation times or the equivalent of eight days for a human cell)," said Rosenberg. The cells had become senescent, meaning they were alive but could not reproduce.

"This was something multicellular animals do, but not unicellular organisms such as E. coli," she said. She and her colleagues are continuing to study that finding.

Funding for this work came from the U.S. Department of Defense Breast Cancer Research Program and the National Institutes of Health.

Rosenberg is a member of the Dan L. Duncan Cancer Center at BCM and the faculty of the BCM Graduate School of Biomedical Sciences. She is also the Cullen Endowed Professor of Molecular Genetics.