December 8, 2014

Structural Snapshots of Damaged DNA

At a Glance

  • Researchers revealed how nucleotides damaged by oxidative stress become inserted into DNA strands and block DNA repair mechanisms.
  • The cell death triggered by this process is thought to play a role in many diseases.
Damaged DNA. After the DNA polymerase (gray molecule in background) inserts a damaged nucleotide into DNA, the damaged nucleotide is unable to bond with its undamaged partner. As a result, the damaged nucleotide swings freely within the DNA, interfering with the repair function or causing double-strand breaks. These steps may ultimately lead to several human diseases.Bret Freudenthal, NIEHS

DNA forms 2 long strands that wind around each other to form a double helix. The building blocks of the strands are known as nucleotides. Nucleotides are assembled into the strands by machinery that includes the enzyme DNA polymerase.

Nucleotides can be damaged by a chemical process called oxidative stress. Ultraviolet exposure, diet, and chemical compounds in paints, plastics, and other consumer products can cause oxidative stress. When nucleotides are damaged, DNA strands become unstable, which may lead to diseases such as cancer, diabetes, hypertension, cardiovascular and lung disease, and Alzheimer’s disease.

A team led by Dr. Samuel Wilson at NIH’s National Institute of Environmental Health Sciences (NIEHS) set out to determine how DNA polymerase inserts damaged nucleotides when assembling DNA strands. They focused on the commonly oxidized 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGTP), which may pair with either cytosine or adenine. The research was funded in part by NIEHS and NIH’s National Cancer Institute (NCI). Results appeared online on November 17, 2014, in Nature.

The scientists formed crystal complexes of DNA, polymerase, and oxidized nucleotides. They used time-lapse crystallography to capture snapshots at different time points during assembly.

The technique revealed that 8-oxo-dGTP is inserted opposite cytosine in one conformation and opposite adenine in another. With either pairing, the oxidized nucleotide causes a nick—an opening between the 2 DNA strands. This nick blocks the DNA repair machinery.

“When one of these oxidized nucleotides is placed into the DNA strand, it can’t pair with the opposing nucleotide as usual, which leaves a gap in the DNA,” Wilson says. “The damaged nucleotide site is akin to a missing plank in a train track. When the engine hits it, the train jumps the track, and all of the box cars collide.”

Oxidized nucleotides are continually removed by the cell. But when they accumulate, the DNA polymerase adds more of them to the strand. Large numbers of the resulting DNA nicks can be lethal to cells and serve as a jumping off point for the development of disease. A better understanding of how this process works may lead to strategies to manipulate it and help prevent or even treat certain diseases.

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Reference: Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. Freudenthal BD, Beard WA, Perera L, Shock DD, Kim T, Schlick T, Wilson SH. Nature. 2014 Nov 17. doi: 10.1038/nature13886. [Epub ahead of print]. PMID: 25409153.

Funding: NIH’s National Institute of Environmental Health Sciences (NIEHS) and National Cancer Institute (NCI); U.S. Department of Energy; Philip Morris USA Inc.; and Philip Morris International.