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Study helps clarify components of DNA ‘copy machine’

November 13, 2015 | Leigh MacMillan

Huzefa Dungrawala and David Cortez

Huzefa Dungrawala, Ph.D., left, David Cortez, Ph.D., and colleagues are studying new proteins involved in DNA replication. (photo by Susan Urmy)

Vanderbilt investigators have generated a “parts list” for the molecular machinery that duplicates DNA each time a cell divides. They were surprised to identify new proteins among the many that participate in copying DNA.

“Imagine that; we’ve been studying DNA replication for 50 years, and we’re still finding new proteins that are required for replication,” said David Cortez, Ph.D., Ingram Professor of Cancer Research and professor of Biochemistry.

The research, published in the journal Molecular Cell, furthers understanding of the DNA “copy machine,” and has implications for cancer therapies that target components of this machinery.

Cortez and his colleagues were particularly interested in understanding the role of a protein called ATR, which responds to DNA damage and is required for replication.

They knew that “when the DNA copy machine encounters a problem, like a paper jam, it stalls. Then ATR comes in and makes sure that the copy machine starts again,” Cortez said.

“Every round of DNA replication requires this DNA damage response; otherwise replication fails and cells die or accumulate lots of mutations and chromosomal abnormalities that lead to cancer,” Cortez said.

Cortez and his team wanted to understand how ATR ensures that DNA replication happens faithfully. One of the prevailing models was that ATR stabilized the stalled machinery — and kept it from falling apart — but the evidence for this was not strong because there was no way to study the copy machine’s “parts,” Cortez said.

Three years ago, Cortez and graduate student Bianca Sirbu developed a method they named iPOND (isolation of proteins on nascent DNA) to examine the replication and damage response machinery. Postdoctoral fellow Huzefa Dungrawala, Ph.D., improved the method and combined it with mass spectrometry to characterize the machinery in a variety of experimental conditions. Kristie Rose, Ph.D., in the Mass Spectrometry Research Center, was critical to the studies.

Using iPOND with mass spectrometry, the researchers identified 218 proteins required to replicate complex genomes and discovered how ATR works.

“It turns out that ATR doesn’t stabilize the copy machine; all the models people have been drawing in the past are wrong,” Cortez said. “ATR still has to undo the paper jam, but it does that by regulating the proteins that come in to fix the jam — not by keeping the copy machine from falling apart.”

Understanding DNA replication is a complex problem, Cortez said.

“There are more than 6 billion base pairs (units of DNA) in every cell that have to be replicated, and that has to happen trillions of times during our lifetime,” he said. “When it doesn’t happen correctly, you get a mutation, you get disease. Our studies tell us something about how replication happens faithfully.”

The proteins they identified as part of the copy machine are “worth paying attention to,” Cortez said. “This project really opened up lots of new things we and others can do.”

The research also offers insight to cancer therapeutics.

ATR is a target for anti-cancer drugs, because cancer cells require this “problem-fixer” more than normal cells, Cortez said.

An inhibitor of ATR is currently in clinical trials.

“Understanding how ATR inhibitors work could tell us which patients might benefit from these drugs or about biomarkers that indicate whether or not the drugs are working,” Cortez said. “It should make us better able to use these drugs in the clinic.”

The research was supported by grants from the National Institutes of Health (CA102729) and the Breast Cancer Research Foundation.