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Nobel Prize honors DNA repair pioneers: three scientists who described the mechanisms that protect our genetic information have been awarded the Nobel Prize in Chemistry.

Take a deep breath. It may not feel any different to you than the other approximately 28,000 breaths you will take today and every other day. But, there is a strong possibility the oxygen you just breathed in caused a cell in your body to suffer a DNA double-stand break. And it may not be that cell's only break. In fact, every one of the 70 trillion cells in the human body suffers one to 10 DNA breaks on a daily basis.

Given that fact, how do humans boast a life expectancy of 78 years if our genetic material--the very essence of who we are--is being ripped apart inside us on a regular basis?

The answer to that question is DNA repair mechanisms. We know this because of three scientists whose work in the field of DNA repair revolutionized our understanding of how living cells function and the molecular-level processes that enable genetic repair.

Tomas Lindahl, Aziz Sancar and Paul Modrich were awarded the 2015 Nobel Prize in Chemistry last month for having mapped and explained just how the cell repairs its DNA and safeguards our genetic information. Here are their stories.

Tomas Lindahl and base excision repair

Working with the RNA molecule as a postdoc at Princeton University in the 1960s, Lindahl began to question molecular stability. The RNA molecule he was working with was very sensitive, especially to heat. If that was the case, how did RNA's cousin, DNA, remain stable for a lifetime?

A few years later, Lindahl would return to his home country of Sweden and the Karolinska Institute in Stockholm to find an answer to that question. His hypothesis was that there are thousands of devastating injuries to the genome every day, so there must be molecular systems for repairing all the DNA defects. And so started 35 years of DNA repair research, ultimately culminating in a Nobel Prize.

Both bacterial and human DNA consists of nucleotides with the bases adenine, guanine, cytosine and thymine. One chemical weakness in DNA is that cytosine easily loses an amino group, which can lead to the alteration of genetic information. In DNA's double helix, cytosine always pairs with guanine, but when the amino group disappears, the damaged remains tend to pair with adenine--which can cause a mutation if the defect persists.

Lindahl focused his efforts there, slowly piecing together the molecular puzzle.

In 1974, Lindahl published his findings that identified uracil-DNA glycosylase (UNG) as the bacterial enzyme that powers base excision repair (BER) functions.

In what the Royal Swedish Academy of Sciences (RSAS) calls a "now-classic study," Lindahl identified UNG in E. coli DNA as the first repair protein, and two years later a second glycosylase, specific for 3-methyladenine DNA. The identification of UNG relied on careful analysis of enzymatic release of uracil as a free base from DNA in vitro.

While Lindahl could already outline the basic principles of BER in his 1974 paper, continued studies led him to reconstitute the entire BER process with purified enzymes from both E. coli and human cells.

RSAS describes the BER process thusly:

"The process is initiated when a DNA glycosylase recognizes and hydrolytically cleaves the basedeoxyribose glycosyl bond of a damaged nucleotide. Once a damaged nucleotide has been identified, the DNA glycosylase kinks the DNA and the abnormal nucleotide flips out. The altered base interacts with a specific recognition pocket in the glycosylase and is released by cleavage of the glycosyl bond. The DNA glycosylase itself often remains bound to the abasic site until being replaced by the next enzyme in the reaction cycle, the apurinic/apyrimidinic (AP) endonuclease, which cleaves the DNA backbone at the S' side of the abasic position. The AP endonuclease also associates with DNA polymerase [beta] (pol-[beta]), to fill the gap. In addition, pol-[beta] harbors a lyase activity, which excises the S'-terminal basefree sugar phosphate residue in a nonhydrolytic elimination process. However, the repair of oxidatively damaged nucleotides has no requirement for such a pol-[beta]-associated lyase activity, because the DNA glycosylases concerned possess endogenous AP lyase activity themselves. In a final step, DNA ligase III/XRCC1 heterodimer interacts with pol-[beta], displaces the polymerase, and catalyzes the formation of a new phosphodiester bond."

Today, thanks in part to Lindahl, we know that BER corrects many different defects that affect the bases, without causing permanent structural damage to the overall DNA strand. To date, more than 100 different types of oxidative lesions have been identified and BER corrects the vast majority of them.

Aziz Sancar and nucleotide excision repair

Sancar is responsible for mapping the precise molecular mechanisms underlying nucleotide excision repair (NER) using the very limited analytical tools of 1970s molecular biology.

Prior work in the field of DNA had found that visible blue light could revive and stimulate growth in bacteria that are exposed to deadly doses of DNA. This became known as photoreactivation, and it alluded to the existence of a light-dependent cellular mechanism that could correct UV-induced damage.

In 1976, working at the University of Texas, Sancar succeeded in cloning the photolyase gene that repairs UV-damaged DNA, and was able to get bacteria to over-produce the enzyme. While substantial, other work in the field overshadowed Sancar's accomplishments thus far. To continue his work, Sancar became a laboratory technician at the Yale University School of Medicine.

By then, it was clear that bacteria have two systems for repairing UV damage--photolyase and a second system that functions in the dark. Sancar's new colleagues at Yale had studied this dark system since the mid-1960s, using three UV-sensitive strains of bacteria that carried three different genetic mutations: uvrA, uvrB and uvrC. However, the bacteria's specific role in NER could not be examined in detail due to the lack of purified proteins available for research purposes in the 1970s.

Sancar had to overcome the limits of current technology to somehow, someway find a technological mechanism to continue his DNA repair research. Ever the pioneer, Sancar developed the Maxicell technique, which relies on a UV-repair deficient bacterial strain.

"After transformation of a plasmid DNA of interest, the hypersensitive bacteria can be UV-irradiated, which causes breakdown of the larger chromosomal DNA, whereas the plasmid molecules that have not been hit by UV can continue to replicate and express proteins. In combination with radioactive amino acid incorporation, the technique allows for labeling and detection of plasmid-encoded proteins in the absence of a chromosomal background," described RSAS in a press release.

The Maxicell technique was then applied to a variety of protein identification projects and allowed Sancar to rapidly identify the proteins encoded by the uvrA, uvrB and uvrC genes.

In 1983, Sancar used the purified uvrA, uvrB and uvrC proteins to reconstitute essential steps in the NER pathway.

RSAS describes Sancar's NER process as such:

"The incisions were performed at four precise locations relative the UV adduct, one at the 8th phosphodiester bond 5' to the lesion and a second at the 4th or 5th bond 3' to the same lesion, thus generating a 12-13 nt long fragment. Later, Sancar could show that the rate of the reaction is stimulated by UvrD (DNA helicase II) and DNA polymerase I (Pol I), which catalyses the removal of the incised strand and synthesis of the new DNA strand, respectively. Finally, DNA ligase catalyses the formation of two new phosphodiester bonds and thus seals the sugar-phosphate backbone."

Of course, Sancar's initial interest in DNA repair was piqued by photolyase. He eventually returned to this enzyme, uncovering the mechanism responsible for reviving the bacteria. In addition, he helped to demonstrate that a human equivalent to photolyase helps us set our circadian clock.

Thanks to Sancar's work, we know today that NER can recognize numerous types of lesions that interfere with normal base pairings and distort the helical structure of DNA. This repair technique corrects problems with its cut-and-patch mechanism.

Paul Modrich and mismatch repair

Although the body's cells are very efficient when it comes to DNA repair, there is always the possibility that an incorrect nucleotide is introduced during synthesis of a new strand. As a result, a non Watson-Crick base pair is formed, which distorts the double-stranded DNA helix. These types of errors are known as mismatches.

Toward the end of the 1970s, Modrich teamed up with Matthew Meselson, a molecular biologist at Harvard University who was studying mismatching bases in DNA. Working together, Modrich and Meselson created a virus with a number of mismatches in its DNA. Since Modrich's previous research focused on the enzyme dam methylase, the researchers used this to add methyl groups to one of the DNA strands.

When the constructed viruses attacked the bacteria, the bacteria consistently corrected the DNA strand that lacked methyl groups. Modrich's conclusion was that DNA mismatch repair is a natural process that corrects mismatches that occur when DNA is copied, recognizing the defect strand by its unmethylated state.

In 1989, Modrich published a paper after he finally reconstituted DNA mismatch correction in a defined in vitro system.

In the paper, according to RSAS, "Modrich demonstrated the requirement of DNA polymerase III, exo-nuclease I, and DNA ligase for mismatch repair. He then combined these factors with purified MutH, MutL, MutS, UvrD, and single-stranded DNA-binding protein. Together these factors could process mismatches in vivo in a strand-specific manner directed by the single, GATC sequence methylated on only one strand (hemimethylated) and located distant from the mismatch."

Later studies by Modrich and others demonstrated mismatch repair in eukaryotic cells, and in 2004 Modrich reconstituted human mismatch repair with only purified factors.

We now know that all but one out of a thousand errors that occur when the human genome is copied are corrected by mismatch repair. However, in human mismatch repair, we still do not know how the original strand is identified. DNA methylation has other functions in our genome to that of bacteria, so something else must govern which strand gets corrected--but exactly what is still a mystery.

Repair systems' role in disease

Every day, these three repair mechanisms, plus several others, work continuously inside the human body to fix thousands of occurrences of DNA damage caused by both internal and external factors--the sun, cigarette smoke, radiation, reactive molecules, etc.

Without these repair mechanisms, our genome would collapse. If just one component fails, the genetic information changes rapidly and the risk of cancer increases.

For example, damage to the NER process causes individuals to become extremely sensitive to UV radiation, often resulting in the development of skin cancer after exposure to the sun. Defects in the mismatch repair process increase the risk of hereditary colon cancer.

Researchers report that in many forms of cancer, one or more of these critical repair systems have been partially or entirely switched off. This makes the cancer cells' DNA unstable, which is one reason why the cells mutate and become resistant to chemotherapy in some instances. However, according to RSAS, the unstable cancer cells are also dependent on the repair systems that are still functioning. Without the remaining, functioning repair systems, the DNA will eventually become too damaged and the cells will die.

Researchers are targeting this weakness as one pathway in the development of new cancer drugs. Inhibiting a remaining repair system allows researchers to slow down or completely stop the growth of cancer.

Lindahl, Sancar and Modrich were awarded the 2015 Nobel Prize in Chemistry for their groundbreaking research in the field of DNA repair. These researchers described the previously indescribable, altering the future of chemistry and science. They provided knowledge about the molecular causes of several diseases, and about mechanisms behind cancer development.

Their research may also make you think twice next time you take a deep breath.

Michelle Taylor, Editor-in-Chief
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Author:Taylor, Michelle
Publication:Laboratory Equipment
Article Type:Cover story
Date:Nov 1, 2015
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