Up until now, Scientists have discovered ten DNA repair pathways, one of which was the object of this year’s Nobel prize in chemistry. In this perspective, Nature just published a piece showing the existence of yet another DNA repair pathway (via the AlkD enzyme), one that is closely related to the repair mechanisms of soil bacteria enzymes. (1) (See also Exhibit A) This discovery has clinical relevance because these enzymes could be reducing the effectiveness of drugs designed to kill cancer cells, thereby further aggravating the “chemoresistance” dilemma.
The newly discovered DNA repair enzyme is a DNA glycosylase, a family of enzymes discovered by Tomas Lindahl, who received this year’s Nobel prize for recognizing that these enzymes removed damaged DNA bases through a process called base-excision repair. (2)
NEW MECHANISMS OF DNA REPAIR: RECOGNITION, REPARATION AND REMOVAL
Contrarily to the base excision repair mechanism, the discoverers found that this enzyme does not require base flipping to recognize damaged DNA or repair it. (3) The discoverers have determined that AlkD enzyme forms a series of interactions with the DNA backbone at and around the lesion while the damage is still embedded within the double helix. Several of these interactions are contributed by three amino acids in the enzyme that catalyze excision of the damaged base. Its characteristics are as follows:
1. Recognition of damaged bases indirectly by identifying lesions without contacting the damaged base itself.
2. Reparation of many different types of lesions as long as they are positively charged. (4)
3. Removal of much bulkier lesions than other glycosylases. (Base excision repair is generally limited to relatively small lesions). (5).
When the structure of DNA was first discovered, (6) scientists thought it was chemically stable. Since this time, biologists have learned that the double helix is in fact a highly reactive molecule that is constantly being damaged and that cells must make unceasing repair efforts to protect the genetic information that it contains. If DNA were too reactive then it wouldn’t be capable of storing genetic information. But then, if it were too stable, it wouldn’t allow organisms to evolve and epigenetics would be science fiction.
Given the continued onslaught of DNA damage that comes from endogenous sources (7) and from modern societies and the environment, including, but not limited to ultraviolet light, toxic chemicals and ionizing radiation, medical science and cancer research must find both innovative and holistic means to better promote the expression of as many DNA repair mechanisms and pathways as possible. (8)
More than 10,000 DNA damage events occur each day in every cell of the human body that must be repaired for the DNA to function properly. Because many cancer drugs destroy and alter DNA within cancer cells, these malignant cells often find ways to repair said damages, thus aggravating the “chemoresistance” problem. It therefore makes sense to combine innovative and holistic techniques to encourage the body’s innate intelligence to better address DNA reparation.
PRECISION AND REFERENCE NOTES
(1). Elwood A. Mullins, Rongxin Shi, Zachary D. Parsons, Philip K. Yuen, Sheila S. David, Yasuhiro Igarashi, Brandt F. Eichman. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions. Nature, 2015; DOI: 10.1038/nature15728
(2) In base-excision repair, 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 pristine base.
(3). Seven years ago, Eichman’s group discovered that AlkD had a structure unlike any of the other glycosylases. The researchers determined that the enzyme was able to locate damaged DNA that has a positive electrical charge. This is the signature of alkylation, attaching chains of carbon and hydrogen atoms of varying lengths (methyl, ethyl etc.), to specific positions on the damaged base. Positively charged alkylated bases are among the most abundant and detrimental forms of DNA damage. However, they are highly unstable, which has made them very difficult to study.
Now the researchers have captured crystallographic sn
4). By contrast, the base-flipping mechanism used by other glycosylases relies on a relatively tight binding pocket in the enzyme, so each glycosylase is designed to work with a limited number of lesions.
(5). A different pathway, called nucleotide excision repair, handles larger lesions like those caused by UV radiation damage. However, Eichman’s team discovered that AlkD could excise extremely bulky lesions, such as the one caused by the antibiotic yatakemycin, which is beyond the capability of other glycosylases.
(6). By Watson and Criket. (2). The DNA double-helix has a spiral staircase structure with the outer edges made from sugar and phosphate molecules joined by stair steps composed of pairs of four nucleotide bases (adenine, cytosine, guanine and thymine) that serve as the basic letters in the genetic code.
(7). Including but not limited to the cell’s own metabolites (the chemicals it produces during normal metabolism), reactive oxygen species and even water.
(8). This year’s Nobel Prize in chemistry was given to three scientists who each focused on one piece of the DNA repair puzzle.
The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions
Elwood A. Mullins, Rongxin Shi, Zachary D. Parsons, Philip K. Yuen, Sheila S. David, Yasuhiro Igarashi & Brandt F. Eichman
Nature (2015) doi:10.1038/nature15728
Received 22 July 2015 Accepted 18 September 2015 Published online 28 October 2015
Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases1, 2. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket2, 3, 4, 5. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge–dipole and CH–π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials6, 7. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism5. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.