Scientists can generate these breaks by a variety of extraneous methods in the laboratory, but Baylor College of Medicine scientists Dr. Philip Hastings and Dr. Susan Rosenberg wondered how such breaks occur without outside help – and how they occur in resting cells not actively making new copies of their DNA. Many of the fully differentiated cells in the body are maintained in a resting state with no need to replicate.
In a report that appears in the journal Nature Communications, they describe how DNA breaks occur in resting forms of Escherichia coli, the model bacterium that they have used to demonstrate ground-breaking understanding of how stress can induce mutations. Resting means that the cells continue to live but they are not actively replicating themselves.
“Spontaneous breaks are the big deal,” said Rosenberg. “How do they occur when scientists are not re-engineering the cell to cause a break?” Rosenberg is a professor of molecular and human genetics at BCM.
Most models of such breakage involve replication – copying a cell’s DNA to pass on its genetic information to daughter cells. The DNA molecule is designed for this process. It is double-stranded, with each strand running in an opposite direction with the A (adenine) in one strand matching to the T (thymine) in the other and the C (cytosine) matching the G (guanine).
The steps in the process involve enzymes called DNA helicases unzipping the paired strands. The DNA does not have to unzip entirely. An area called the “replication fork” unzips and moves down the whole length of the DNA molecule.
Small proteins called single strand binding proteins temporarily bind to each side to keep them separate. Then DNA polymerase “walks” the DNA strands and adds new nucleotides to each, with the complementary nucleotides bindings to nucleotides on the existing strand (A to T and C to G). The unzipping provides opportunities for DNA breakage.
One way that breaks occur is when an enzyme called RNA polymerase collides with the replication machinery. This happens when the RNA polymerase has “backtracked” on the DNA template, or when RNA and DNA polymerases meet head on (shown previously at BCM by the laboratories of Drs. Christophe Herman and Jue Wang (now at the University of Wisconsin), also faculty in the department of molecular and human genetics). All known means of DNA breakage so far have some role for the replication machinery, yet scientists know that breaks happen in cells that are not replicating DNA.
The question, then, is how do breaks happen when there is no replication?
That question begins with RNA loops, hybrid RNA-DNA structures that their paper shows are the “precursors of mutagenesis,” said Philip Hastings, professor of molecular and human genetics at BCM and corresponding author of the report.
“The role of R loops is to produce double-strand breaks,” said Hastings. He said that these kinds of loops that result from RNA-DNA hybrids are more widespread under stress conditions, such as starvation.
“Under most circumstances, RNA from gene transcription is protected against being incorporated into the DNA,” he said. “However, we are working in starving cells that might not be able to produce enough protein to ensure this kind of protection.”
Mutation hot spots
In previous work, Rosenberg and Hastings have shown that the genome in stressed cells has “hot spots” for mutation. They are beginning to wonder if the cell somehow controls where the mutation occurs by controlling where it makes a double-strand break.
“We don¹t know yet,” said Rosenberg. “But what this new study says is that breaks are more likely to be made in a transcribed region than in an untranscribed region. The genes in use are the ones most likely to be mutagenized.”
Hastings credited Hallie Wimberly, an intern in his laboratory and now a graduate student at Yale University School of Medicine in New Haven, Conn., with doing important work in this report. Others who took part include Chandan Shee, P.C. Thornton and Privya Sivaramakrishan, all of
Funding for this work came from the National Institutes of Health (Grants R01-GM53158, NIH Director’s Pioneer Award DP-CA174424 and R01-GM64022. Rosenberg holds the Ben F. Love Chair in Cancer Research.
For more information on research at Baylor College of Medicine, please go to www.bcm.edu/fromthelab.