Study reveals how enzymes act to protect the genome

FAPESP/DICYT In an article published in the journal Molecular Cell, researchers at Cornell University in the United States describe a new technique that reveals details of the action of enzymes responsible for protecting the genome against problems occurring during the DNA replication process.

The authors include three Brazilians: Marcus Bustamante Smolka and José Renato Cussiol, who are former recipients of doctoral scholarships from FAPESP and are currently at Cornell, as well as Francisco Bastos de Oliveira, who recently earned tenure as an associate professor at the Federal University of Rio de Janeiro (UFRJ).

“This new technique can assist in the development of drugs with more specific actions against different types of cancer and in the understanding of diseases relating to the malfunctioning of enzymes known as kinases that regulate all of the important processes inside cells,” Smolka said.

Kinases are enzymes that catalyze a chemical reaction called phosphorylation, which transfers high-energy phosphate molecules such as ATP (adenosine triphosphate) to target proteins (substrates).

In certain cases, this reaction may lead to activation or inactivation of the target protein or may act as a signal for degradation of the molecule. Thus, kinases regulate processes such as cell division, proliferation and differentiation, among others.

“It’s estimated that between five and ten of the more than 500 kinases described in the human genome play the role of orchestrating cellular defense against DNA replication problems,” Smolka said.

These errors typically occur moments before the cell divides, when the DNA strands in the nucleus unwind so that the genetic code can be copied.

“From the fertilization of an egg cell to the formation of the adult organism, the genome must be replicated over 10 trillion times. The cell requires a system to detect and address defects in the copying process, or else there will be an accumulation of damage to the genome that will make life unviable. Kinases are essential to this damage containment process,” Smolka said.

When damage is detected, the kinases dispatch various rescue teams in an attempt to prevent cell death. “It’s as if there was a huge leak in a city, and the enzymes were responsible for shutting off the water, warning the inhabitants, calling in a repair crew, telling the police to stop the traffic, and so on. However, how this signaling happens wasn’t yet understood.”

In the paper published in Molecular Cell, the Cornell group shows how three of these enzymes that are dedicated to protecting the genome behave. They are the equivalent in yeast to the human kinases ATR, ATM and CHK1.

Considering that a single cell may have more than 20,000 phosphate radicals being simultaneously transferred from one site to another by different kinases, identifying what each of these enzymes does is no trivial task. To conduct this investigation, the researchers used a mass spectrometer, a device that measures molecular mass with very high precision.

“We extracted proteins from yeast cells and broke them down into several pieces. The spectrometer can measure the mass of each piece and identify whether a phosphate group is bonded to it. This enables us to know where the radicals are transferred to and from. The precision is so high that we even know which amino acid in the target protein that the phosphate group bonds to,” Smolka said.

Kinase maps

More than 100 different substrates were identified for the enzyme ATR alone, which attracted the most attention from scientists. To identify these substrates, they created three mutant yeast lineages – one without the ATR gene, another with the ATR gene, and a third without CHK1.

“This technique enabled us to detect more than 6,000 phosphorylation events,” Smolka said. “By comparing a normal cell with one of the mutants, we were able to observe which of these events disappeared and thus to identify the substrate for each enzyme.”

In addition, they developed quantitative maps, christened QMAPS (Quantitative Mass Spectrometry Analysis of Phospho-substrates), to show how the phosphorylation of each substrate changed in different conditions.

“For example, we were able to see whether a drug that damages DNA increases or decreases phosphorylation events in each target protein. In other words, we were able to identify not only when and how ATR is activated but also at what level its different substrates are regulated,” Smolka said.

The researchers were surprised to discover via QMAPS that ATR is only not activated when the genome is damaged. The enzyme also acts preventively.

“ATR is always activated, and rather than signaling to four or five proteins, as expected, it regulates hundreds. The process is far more complex than we anticipated,” Smolka said.

According to Smolka, ATR inhibitors are already being clinically tested against cancer. Because malignant cells divide very rapidly and in a disorderly manner, they depend much more on the action of ATR to survive than do normal cells.

“It’s as if there were lots of leaks throughout the city, but even so, the cancer cell manages to survive,” Smolka said. “QMAPS can help us identify how and in what situations this kinase must be inhibited to kill different types of cancer with minimal interference in normal cells.”

The technique can also be used by other research groups devoted to understanding more about the role of kinases in the human organism as well as in other animals and in plants.

Studies have already shown that these enzymes are important targets for the development of new drugs. It is estimated that of the more than 500 kinases identified in the human genome, fewer than 100 have been thoroughly studied to date.

“At present, we’re engaged in similar experiments to those described in the journal paper but with human lineages. We want to connect the work we’ve done here at Cornell with various interesting initiatives that are happening in São Paulo,” Smolka said.