Throughout our lifetimes, from fertilized egg to adult, our cells must divide many times. To do that, cells must copy our whole genome of approximately three billion base pairs every time they divide. Special proteins come together to form a molecular machinery called the replisome, which unwinds the double helix of DNA in a cell, exposing the two strands and synthesizing a new, complimentary sequence of DNA for each.
“Imagine going through billions of these little ladders in just a few hours,” says Marcus B. Smolka, Molecular Biology and Genetics. “The replisome has to make a perfect copy. If it makes a mistake, mutations or chromosomal breakage result, and that is the hallmark of cancer.”
ATR—Monitoring the Replication Process
Many of the mutations that lead to cancer are in genes that make proteins required for replication and maintenance of the genome. Smolka and his lab study a class of proteins, known as kinases, with important roles in genome replication. They have found a particularly fruitful area of research in the kinase ATR, the master protein that monitors the replication process.
“If something goes wrong, ATR can detect it and will come to the region where the problem is,” Smolka explains. ATR is an enzyme, so it can modify other proteins by adding a phosphate to them. This phosphorylation starts a signaling cascade, a circuitry of events that orchestrates the detection and repair of damage before the cell divides in two.
“Before we started looking at this, people thought ATR worked in a linear pathway where it would target one protein, and that protein would target another protein,” Smolka says. “Our work has changed the paradigm of how the action of this kinase is viewed. It’s not a simple action; it’s really a network of events. ATR phosphorylates hundreds of different proteins.”
ATR and Cancer Cells
Understanding ATR’s fundamental role in replication is important for cancer research because cancer cells depend on ATR much more than normal cells do. “Cancer cells proliferate much faster than normal cells, which causes stress in the replication machinery and more chromosomal breakage, so they get addicted to ATR,” says Smolka. “They need it to repair the damage. If ATR is not there, they will die after one replication cycle. Replication in normal cells tends to be much more regulated and robust, so if you inhibit ATR a bit, normal cells will still be fine.”
Smolka’s research sheds light on work done by others in the scientific community who are focused on creating drugs that inhibit ATR. These drugs are currently in clinical trials for cancer treatment. “We’ve studied the action of ATR inhibitors, and they are very powerful,” says Smolka. “The problem is that researchers working on creating these drugs can see their effects—cancer cells die—but they don’t understand how the drugs do what they do. Our work is revealing what processes are being affected by the inhibitors.”
Techniques for Capturing ATR in Action
Part of Smolka’s basic research focuses on the action of kinases in yeast, which is a simple system that has analogous aspects with human biology. “We work in yeast and also in mammalian systems and human cell lines,” he says. “But it’s much easier to map the action of these kinases and come to a fundamental understanding of what they do in yeast. That gives us a powerful place from which to approach the understanding of human kinases.”
Smolka had to develop special techniques to capture the action of ATR and other kinases. To pinpoint ATR’s action in a cell, his lab uses mass spectrometry, which identifies the chemical constitution of a substance by separating ions according to their differing mass and charge.
“We can break open cells and extract pieces that make up the thousands and thousands of proteins,” he says. “To figure out what ATR is doing in cells, we are able to analyze these pieces of proteins and define which have the particular phosphate used by ATR to modify a target. That way we can map precisely where the phosphate is, and we can do it in a quantitative manner. That allows us to determine exactly what proteins ATR is targeting.”
Key Discoveries for Understanding Cancer
A key question Smolka and his colleagues want to answer centers on how ATR maintains the integrity of the genome. In 2017 they discovered that kinase promotes repair of chromosomal breaks in DNA. Kinase controls proteins that carry out the repair through the process of homologous recombination, where nucleotide sequences are exchanged between similar or identical molecules of DNA. “That was a very exciting finding,” Smolka says. “I think it will allow us to define the mechanism for how ATR works to prevent breakage to chromosomes.”
The researchers made another unexpected discovery that may also be a game changer for understanding cancer. They found that ATR can act in very different ways, depending on how it gets activated. When ATR is activated at a cell’s DNA break, it promotes DNA repair; when it is activated at other locations in the cell, it does not promote repair, but instead, it allows cells to replicate faster.
“This was a new function for ATR that we didn’t know about before. It could help explain why cancer cells are addicted to ATR.”
“This was a new function for ATR that we didn’t know about before,” Smolka says. “It could help explain why cancer cells are addicted to ATR. This kinase not only prevents chromosomal breakage, but it allows the replisome to go faster through the DNA so that cells replicate more quickly. We’re trying to figure out right now how it does that.”
Effective Cancer Therapies—Possible through Mapping the Action of Kinases
ATR is just one of many kinases the Smolka Lab studies. The researchers are turning their attention to several others in the VRK and TLK families that also appear to affect cancer cell replication. They are also continuing to branch out and improve the technology they use to map the action of kinases. They have begun using a genetic editing system known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) that uses the nuclease Cas9 to target specific DNA sequences. “We are creating human cell lines where we can remove specific kinases,” Smolka explains. “We can then look at what set of target phosphates also disappear. That way we can map what each kinase does.”
The more scientists understand about the mechanisms behind how kinases work, the more applications for cancer therapy should result. To understand how cells replicate and maintain their stability—and why cancer cells are so good at it—the action of kinases must be understood from a more integrated perspective. “There are a lot of biology questions that are still unanswered,” Smolka says. “I think there is a huge potential to bring in new technology to understand these complicated systems from a holistic perspective and at the same time to bring the research down to a reductionist view and pinpoint a specific event. Our lab has the uncommon ability to do both these things.”