Scientists working with yeast at Duke University School of Medicine say they have shown that chromosomal fragile sites appear in specific areas of the genome where the DNA-copying machinery is slowed or stalled, either by certain sequences of DNA or by structural elements. The researchers believe their findings could provide insights into the origins of many of the genetic abnormalities seen in solid tumors.
“Other studies have been limited to looking at fragile sites on specific genes or chromosomes,” said Thomas D. Petes, Ph.D., the Minnie Geller professor of molecular genetics and microbiology at Duke University School of Medicine. “Ours is the first to examine thousands of these sites across the entire genome and ask what they might have in common.”
The term “fragile sites” was first coined in the 1980s to describe the chromosomal breaks that appeared whenever DNA polymerase was blocked in mammalian cells. Since that discovery, research in Saccharomyces cerevisiae has shown that certain DNA sequences can make the polymerase slow down or pause as it makes copies. However, none of them have shown how those delays result in fragile sites.
In the Duke study (“Genome-wide high-resolution mapping of chromosome fragile sites in Saccharomyces cerevisiae”), which appears in the Proceedings of the National Academy of Sciences, Dr. Petes wanted to find the link between the copier malfunction and its genetic consequences on a genome-wide scale. First, he knocked down the levels of DNA polymerase in yeast cells to ten-fold lower than normal. Then he used microarray technology to map where segments of DNA had been rearranged, indicating that a fragile site had once been there.
After finding those fragile sites, his laboratory spent more than a year combing through the literature for any recurring themes among the genomic regions they had uncovered. Eventually they showed that the fragile sites were associated with sequences or structures that stalled DNA replication, entities such as inverted repeats, replication termination signals, and transfer RNA genes.
“We also show that low levels of DNA polymerase greatly elevate the frequency of deletions and duplications (reflecting unequal sister-chromatid recombination between repeated genes), and result in changes in chromosome number (aneuploidy),” wrote the investigators.
“The ability to analyze these sites on a genome-wide basis is an important advance,” said Gray Crouse, Ph.D., an expert unaffiliated with the new study who is a professor of biology at Emory University. “It has been known for a long time that many cancer cells have an abnormal number of chromosomes, and many different chromosome rearrangements have been observed in various tumor cells. It is likely that there are many different causes of chromosome instability in cancer cells. The current work suggests that those chromosomal rearrangements observed at fragile sites and found in solid tumors may be due to breaks from perturbed replication.”