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Scientists in the OHSU School of Medicine’s Department of Biochemistry and Molecular Biology are the first to report a new approach using eggs of the African clawed frog, which goes by the Latin name Xenopus laevis, to understand how the Fanconi anemia proteins ensure that DNA is replicated properly, according to a study published this month in the journal Molecular and Cellular Biology.

The international team is led by the OHSU laboratory of Maureen Hoatlin, Ph.D. Using extracts from Xenopus eggs and chemically triggering DNA copying, the team showed that the Fanconi proteins function to prevent accumulation of breaks in DNA strands that arise even during normal replication. Fanconi anemia is thought to be the result of a defect in the Fanconi genes’ ability to repair DNA damage.

Hoatlin said there are many advantages to using the frogs’ eggs, instead of human cells, to study Fanconi anemia.

“In human cells, most of the Fanconi proteins are hard to detect, so you have to grow millions of cells over long periods of time to collect enough to study,” she said. “The other problem is that cultured human cells are at all different stages of the cell cycle. The bulk of the cells are not rapidly dividing, and it’s only when cells are dividing that the Fanconi proteins are usually at their highest expression and activity.”

In Xenopus eggs, however, Fanconi proteins are stockpiled in preparation for the rapid divisions that occur after fertilization. Plus, the divisions occur at the same time, or synchronously, allowing naturally regulated stages of division to be studied simultaneously.

“It’s very hard to synchronize mammalian cells,” Hoatlin added. “Also, unfortunately for Fanconi researchers, methods used to synchronize cells are usually also the ways you might damage the DNA in cells, whereas this system allows us to look at unperturbed replication.”

The use of Xenopus eggs as a cell-free system for studying checkpoints in the DNA damage pathway is nothing new. Since the mid-1980s, scientists have been using the BB-sized, black-and-white, bead-like eggs to probe orchestration of the DNA replication process, something not as easily done in mammalian cells.

Since a Xenopus egg isn’t fertilized until after it is laid outside the body of the female frog, it’s in a relative state of suspension until it is fertilized. During this time, when the cell cycle is arrested, scientists can use chemicals and a centrifuge machine to keep its DNA from replicating. This creates a convenient extract rich in all the essential components ready for full replication.

Then, by chemically activating the extract and adding sperm DNA from the male frog, the proteins in the extract unwind the DNA and the replication process is off and running under the watchful eyes of the researchers.

“That’s just the beginning of what we want to do with these extracts to study how the Fanconi proteins work,” Hoatlin said. “You can control the extract with chemicals or by removing proteins with specific antibodies. It’s a very powerful system.”

In the Molecular and Cellular Biology paper, Hoatlin’s team identified many of the Xenopus versions of the Fanconi genes and found that they were very similar to the human counterparts by sequence and behavior. For example, the Fanconi proteins were drawn to the DNA once the copying process began. By adding a protein called geminin to the extract, which prevents the assembly of protein complexes essential to the beginning steps of replication, the scientists found the Fanconi proteins no longer accumulated on the DNA, even if the DNA was damaged.

The implication, Hoatlin pointed out, is that even if the DNA is damaged, “unless there’s replication, the Fanconi proteins don’t recognize the damage.”

“We wanted to develop an approach that would allow us to determine how the Fanconi proteins assemble on replicating DNA and what proteins control the important steps. These extracts give us the tool we need for those experiments,” she said. In fact, in the new Molecular and Cellular Biology study, Hoatlin’s lab already found that an important regulator of the cell’s DNA damage-sensing mechanism controls some, but not all, of the Fanconi protein’s arrival on replicating DNA.

Stacie Stone, an OHSU graduate student and co-lead author of the study, believes Hoatlin’s lab, where she has worked with frogs for more than two years, will continue to yield discoveries that may someday lead to a treatment of certain cancers and for Fanconi anemia, a devastating disease that primarily affects children. The lab already joined forces with other labs to isolate several of the dozen genes known to exist in the Fanconi pathway.

“Really, there are not a whole lot of labs using the Xenopus extract approach yet,” Stone said. “There are so many experiments we’d like to do now.” For example, Alexandra Sobeck, Ph.D., the study’s other co-lead author and a scientist in Hoatlin’s lab, is examining the order of assembly of Fanconi proteins on DNA and how the Fanconi proteins are activated in response to DNA damage.

“What I’m seeing is that Fanconi proteins work together with other important protein complexes,” Sobeck added. “Every day is exciting.”

SOURCE:: OHSU News