The new type of DNA repair enzyme, AlkD on the left, can identify and remove a damaged DNA base without forcing it to physically “flip” to the outside of the DNA backbone, which is how all the other DNA repair enzymes in its family work, as illustrated by the human AAG enzyme on the right. The enzymes are shown in grey, the DNA backbone is orange, normal DNA base pairs are yellow, the damaged base is blue and its pair base is green. [Brandt Eichman, Vanderbilt University]
Hot on the heels of the recent announcement of the Nobel Prize in Chemistry being awarded for seminal discoveries in the area of DNA repair, researchers at Vanderbilt University have published data describing new enzymatic activity for a DNA glycosylase discovered previously in the bacteria Bacillus cereus.
When Watson and Crick first published their now famous double-helix structure of DNA, many scientists imagined the molecule to be extremely chemically stable—acting as the template for passing along inheritable genetic traits. However, over the years investigators have since discovered DNA’s susceptibility to damage and its dynamic nature to repair itself, to maintain genomic stability.
“It's a double-edged sword,” remarked senior author and project leader Brandt Eichman, Ph.D., associate professor of biological sciences and biochemistry at Vanderbilt. “If DNA were too reactive then it wouldn't be capable of storing genetic information. But, if it were too stable, then it wouldn't allow organisms to evolve.”
There are many ways that DNA can become damaged, but they can be classified into two basic groups: environmental sources including ultraviolet light, toxic chemicals, and ionizing radiation and internal sources, which include, reactive oxygen species, a number of chemicals the cell produces during normal metabolism, and even water.
“More than 10,000 DNA damage events occur each day within every cell of the human body, which must be repaired for DNA to function properly,” explained lead author Elwood Mullins, Ph.D., a postdoctoral research associate in Dr. Eichman’s laboratory.
The Vanderbilt team discovered the new repair activity while studying the DNA glycosylase AlkD. Glycosylases are part of a family of enzymes discovered by Tomas Lindahl, Ph.D., who received this year's Nobel prize for recognizing that these enzymes removed damaged DNA bases through a process called base-excision repair (BER).
Briefly, during BER, a specific glycosylase molecule binds to DNA at the location of a lesion and bends the double-helix in a way that causes the damaged base to flip from the inside of the helix to the outside. The enzyme fits around the flipped out base and holds it in a position that exposes its link to the DNA's sugar backbone, allowing the enzyme to detach it. After the damaged base has been removed, additional DNA-repair proteins move in to replace it with a new, undamaged base.
Dr. Eichman and his team found that AlkD from B. cereus works in a totally different fashion—as it does not require base flipping to recognize damaged DNA or repair it. Using crystallography techniques, the researchers were able to determine that AlkD forms a series of interactions with the DNA backbone at and around the lesion while the lesion is still stacked in the double helix. Several of these interactions are contributed by three amino acids in the enzyme that catalyze excision of the damaged base.
The findings from this study were published recently in Nature through an article entitled “The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions.”
Additionally, the investigators found that AlkD identifies lesions by interacting with the DNA backbone without contacting the damaged base itself and can repair many different types of lesions as long as they are positively charged. Since the enzyme doesn’t have the same type of binding pocket, it isn't restricted in the same way as other glycosylases. Lastly, AlkD can excise much bulkier lesions than other glycosylases. Base excision repair is limited to relatively small lesions. A different pathway called nucleotide excision repair typically handles larger lesions like those caused by UV radiation damage. However, Dr. Eichman's team discovered that AlkD could excise lesions that would normally default to other DNA repair pathways.
“Our discovery shows that we still have a lot to learn about DNA repair and that there may be alternative repair pathways yet to be discovered. It certainly shows us that a much broader range of DNA damage can be removed in ways that we didn't think were possible,” Dr. Eichman stated. “Bacteria are using this to their advantage to protect themselves against the antibacterial agents they produce. Humans may even have DNA-repair enzymes that operate in similar fashion to remove complex types of DNA damage. This could have clinical relevance because these enzymes if they exist, could be reducing the effectiveness of drugs designed to kill cancer cells by shutting down their ability to replicate.”
