When chromosomes experience double-strand breaks resulting from oxidation, ionizing radiation, replication errors and certain metabolic products in cells, they utilize their genetically similar chromosomes to patch the gaps via a mechanism that involves both ends of the broken molecules. To repair a broken chromosome that lost one end, a unique configuration of the DNA replication machinery is deployed as a desperation strategy to allow cells to survive, the researchers discovered.
The collaborative work of graduate students working under Anna Malkova Ph.D., associate professor of biology at Indiana University-Purdue University Indianapolis (IUPUI) and Kirill Lobachev, Ph.D., associate professor of biology at the Georgia Institute of Technology, was critical in the advancement of the project.
The group’s research will be published online this week in the Nature journal, with two graduate students (Natalie Saini from the Georgia Institute of Technology and Sreejith Ramakrishnan from the School of Science at IUPUI) as first authors. Other collaborators include Dr. James Haber, Ph.D., Brandeis University, and Grzegorz Ira, Ph.D., Baylor College of Medicine.
“Previously, we have shown that the rate of mutations introduced by break-induced replication is 1000 times higher as compared to the normal way that DNA is made naturally, but we never understood why,” Malkova said.
The latest research reveals a mode of replication that can operate in non-dividing cells—the state of most of the body’s cells—making this kind of replication a potential route for cancer formation.
“Potentially, this is a textbook discovery,” Lobachev said.
The two labs used cutting-edge analysis techniques and equipment available at only a handful of facilities around the world. This allowed the researchers to see inside yeast cells and freeze the break-induced DNA repair process at different times. They found this mode of DNA repair doesn’t rely on the traditional replication fork — a Y-shaped region of a replicating DNA molecule — but instead uses a bubble-like structure to synthesize long stretches of missing DNA. This bubble structure copies DNA in a manner not seen before in eukaryotic cells and leads to conservative DNA synthesis that promotes highly increased mutagenesis.
Traditional DNA synthesis, performed during the synthesis-phase of the cell cycle, is done in a semi-conservative manner as proven by Matthew Meselson and Franklin Stahl in 1958, shortly after the discovery of the DNA structure. They found two, new double helices of DNA are produced from a single DNA double helix, with each new double helix containing one original strand of DNA and one new strand. This experiment was termed “the most beautiful experiment in biology.”
“From the point of view of the cell, the whole idea is to survive, and this is a way for them to survive a potentially lethal event. But, it comes at a cost,” Lobachev said.
During break-induced replication, one broken end of DNA is paired with an identical DNA sequence on its partner chromosome. Replication that proceeds in an unusual bubble-like mode then copies hundreds of kilobases of DNA from the donor DNA through the telomere at the ends of chromosomes.
“The break-induced replication bubble has a long tail of single-stranded DNA, which promotes mutations,” Ramakrishnan said. “The single-stranded tail might be responsible for the high mutation-rate, because it can accumulate mutations by escaping the other repair mechanisms that quickly detect and correct errors in DNA synthesis.”
“This is a way of synthesizing DNA in a very robust manner,” Saini added “The synthesis can take place and cover the whole arm of the chromosome, so it’s not just some short patches of synthesis.”
“This is a way to essentially mutagenize the genome that is not supposed to be replicating,” Lobachev said.
When it comes to cancer, other diseases and even evolution, what seems to be happening are bursts of instability, and the mechanisms promoting such bursts were unclear, Malkova said. The molecular mechanism of break-induced replication unraveled by the new study provides one explanation for the generation of mutations.