Genetic Breaking Point
Researchers show slowed gene replication leads to chromosome fragility

Researchers have long hypothesized that slow or altered gene replication leads to chromosome fragility, but have never been able to prove it. Now two Tufts molecular biologists have found a highly flexible DNA sequence that increases fragility and stalls gene replication, leading the chromosome to break. That’s important because many kinds of cancer are associated with areas in the human genome where chromosomes break.

Using a genetic test in yeast, Catherine Freudenreich, an associate professor of biology, and doctoral student Haihua Zhang focused on a specific—and common—fragile site that lies in the middle of a tumor suppressor gene. Chromosome breakage in the area is strongly associated with cancer, since absence of the tumor suppressor gene can lead to tumor growth. “The fact that there is fragility in the same region that this gene is located in is a bad coincidence,” Freudenreich says. “Fragility can cause deletions [in the genes], and deletions can cause cancer, so you want to understand the fragility, because that might be what’s causing cancer.”

“You want to understand the fragility, because that might be what’s causing cancer,” says Catherine Freudenreich, associate professor of molecular biology.

Prior research had predicted flexibility of the DNA in this particular site, suggesting that the flexibility was connected to the fragility. To test the theory, Freudenreich and Zhang used yeast chromosomes, which allowed them to monitor the replication process and to study the region in a more detailed way than they could by examining human chromosomes. They expect the results will be similar when tested in human cells.

The researchers took two regions of predicted high flexibility, plus a region near a cancer cell breakpoint and a control region, and tested whether any of those regions could cause breakage of a yeast chromosome. “We found that one did,” says Freudenreich. “This is exciting because it is the first known sequence element within a human common fragile site shown to increase chromosome breakage. What is intriguing is that the sequence that breaks, in addition to being flexible, is predicted to form an abnormal DNA structure.”

Next, the researchers had to determine how the chromosomes were breaking. From past studies, they hypothesized that breakage was connected to gene replication. As cells divide, the DNA inside those cells must make copies of themselves, and it turns out that the chromosomes were breaking because replication was stalled. “We found that the fragile sequence actually stops replication,” says Freudenreich. “So when replication gets there, it has trouble, it stops, it pauses, it can’t go further very easily.”

Most of the time, chromosomes break and heal correctly, but problems arise when they do not. “Cancer cells almost always have some sort of deletions or rearrangements,” she said. “Something is wrong with their chromosomes, which then messes up the genes that are in those areas.”

The researchers also noticed that in this particular sequence ,the DNA was composed mostly of the bases adenine (A) and thymine (T), rather than the other bases cytosine (C) or guanine (G). Freudenreich and Zhang, whose paper was published in Molecular Cell, found the longer the AT-repeat, the more the replication process stalled. “We think the longer the repeat, the more the abnormal DNA structure forms, and the more fragile your chromosome is, but we haven’t completely been able to nail that down,” Freudenreich says. “Would people with longer repeats be more prone to deleting that tumor suppressor gene and getting cancer as a result? We have made this correlation, and we’d like to know if it has a medical consequence.”

This story ran in the December 2007 issue of the Tufts Journal.