Researchers at the Cross Cancer Institute in Edmonton, Alberta, have come up with a relatively high-throughput way to analyze proteins that have covalently cross-linked to DNA due to radiation exposure.
The research aims to shed light on the cytotoxic effects of radiation used in the treatment of cancer, explained Sharon Barker, the lead author of a study describing the new method that was published in last week's issue of Analytical Biochemistry.
While previous methods identified only one or two proteins at a time that were crosslinked to DNA, Barker's study identified about 30 DNA-crosslinked proteins. Of those, eight had been found before by other means.
The trick to identifying so many DNA cross-linked proteins in a relatively short period of time was in the protein isolation process, said Barker. While other researchers had previously used filters to isolate DNA that was attached to proteins, Barker and her research team decided to first isolate DNA using a chaotropic agent called DNAzol, or guanadine hydrochloride, that is sold by Invitrogen. They then used salt, urea, and detergent to stringently strip away proteins that were not covalently bound to the DNA.
Finally, mass spectrometry was used to identify the proteins that were left after stripping.
"Certainly, this is the first time that such a large number of [DNA-crosslinked] proteins have been identified."
"Certainly, this is the first time that such a large number of [DNA-crosslinked] proteins have been identified," said Barker. "Others may have isolated large numbers of DNA-crosslinked proteins on 2D gels, but they either didn't have a sufficient amount [of] protein, or didn't have the technology to be able to identify them."
According to Barker, the chaotropic method is more sensitive than older filter trap methods.
"With the filter trap, any sort of handling that shears DNA would result in losing it through the filter," said Barker. "We do feel that our method is more sensitive and yields a purer sample that makes the mass spec easier to do."
Barker said that her team's study differed from previous radiation studies because it focused on "biologically relevant" doses of radiation. Barker and her colleagues exposed hamster and human cells to radiation doses between 0.5 Gy and 2 Gy in strength, then studied the DNA-protein crosslinking in those cells.
"If you're giving someone a treatment for cancer, the standard dose is two Gy," said Barker. "Certainly, we felt it was very important to do this at a biologically relevant dose. We're interested not only in radiation exposure as a carcinogenic, but also radiation used in the treatment of cancer."
The proteins identified by Barker's group included chromatin regulatory proteins, scaffold proteins, and cell stress proteins. For nearly every protein identified, Barker's group was able to confirm in the literature that the protein had a nuclear function.
"We did find a diversity of proteins. The proteins belonged to a number of different functional groups," said Barker. "For example, we found GAPDH, which is usually known as a regulatory housekeeping gene. But when we looked in the literature we found that GAPDH also has some new associations and functions, including DNA repair."
A recent PhD graduate in Michael Weinfeld's laboratory, Barker said she decided to do her doctoral thesis on DNA-protein crosslinking because the phenomenon had not been well studied. While double-strand breaks in DNA caused by radiation have received a considerable amount of attention, DNA-protein crosslinking due to radiation has been virtually ignored, Barker said.
"If you really want to understand the cytotoxic effects of radiation, you need to know what each type of damage is contributing, and there was a dearth of information on DNA-protein crosslinking," said Barker. "If you think about covalently trapping proteins to DNA, there can be a lot of consequences how are you going to do replication or transcription or repair if you've got an anchor?"
Now that she and her team have identified about 30 proteins that covalently cross-link to DNA, Barker said that the next step is to study how those proteins are involved in the repair pathway using hamster cells.
"There are a lot of DNA repair mutants available with hamsters," she explained. "What we're now working on looking at is how each repair pathway is going to handle these types of damages by different crosslinked proteins."
One question that remains is whether the total amount of crosslinked DNA determines the degree of cytotoxicity, or whether the specific type of DNA-crosslinked protein also plays a role in cell damage.
"At this point there are no [crosslinked] proteins that we are particularly interested in more than others, but we did see different patterns of crosslinking," said Barker. "For example, some proteins were induced to crosslink to a greater extent with the presence or absence of oxygen."
The presence or absence of oxygen during radiation exposure is relevant because many tumors have areas of hypoxic cells, or cells that are not exposed to oxygen, Barker explained. Those cells tend to be resistant to radiation.
"We need to know what is different about those cells that's allowing them to survive radiation," said Barker. "With this nice, new stringent method, we can do a protein-by-protein dissection of crosslinking. It opens the door to a lot of new work in this area."
Tien-Shun Lee ([email protected])