Where Smoking Tars DNA, Repair May Be Lacking

Scientists have known for decades that smoking cigarettes causes DNA damage, which leads to lung cancer. Now, scientists are beginning to study exactly where such damage occurs in the genome, and where it is most likely to be repaired. Damage maps have been drawn by scientists using a new method that determines the sequence specificity of the nucleotide excision repair mechanism. [Christ-Claude Mowandza-Ndinga/UNC Health Care]

For smokers, the molecular equivalent of tar-coated lung tissue is adduct-blighted DNA. The adduct, called benzo[α]pyrene diol epoxide (BPDE), is chemical that forms when the body tries to break down benzo[α]pyrene (BaP), a potent carcinogen that is a byproduct of burning organic compounds, and is never more harmful than when it is sucked into the lungs, as smokers do on a regular basis. BPDE sticks to DNA, interfering with its function and potentially causing cancer.

BPDE’s sticky, disease-causing ways are getting a closer look now that technologies are being developed to study DNA repair mechanisms such as nucleotide excision repair, which involves the recruitment of special proteins that perform DNA surgery. They snip out sullied portions of a DNA strand. If all goes well, DNA-synthesizing enzymes then reconstruct the missing section of DNA from that section’s complement, that is, from sequence information provided by the unaffected complementary strand.

New techniques are capable of mapping where this kind of DNA repair occurs. For example, University of North Carolina School of Medicine scientists have developed a general method for genome-wide mapping of nucleotide excision repair at single-nucleotide resolution. These scientists, led by Nobel Nobel laureate Aziz Sancar, M.D., Ph.D., have applied this method to generate repair maps of ultraviolet (UV)- and BaP-induced DNA damage in humans.

Details of the work appeared June 12 in the Proceedings of the National Academy of Sciences, in an article entitled “Human Genome-Wide Repair Map of DNA Damage Caused by the Cigarette Smoke Carcinogen Benzo[a]pyrene.” Although the article focuses on the damage caused by smoking, it emphasizes that the new damage-assessment method can be used to study repair of all types of DNA damage that undergo nucleotide excision repair.

“We have developed a method for capturing oligonucleotides carrying bulky base adducts, including UV-induced cyclobutane pyrimidine dimers (CPDs) and BaP diol epoxide-deoxyguanosine (BPDE-dG), which are removed from the genome by nucleotide excision repair,” the article’s authors wrote. “The isolated oligonucleotides are ligated to adaptors, and after damage-specific immunoprecipitation, the adaptor-ligated oligonucleotides are converted to dsDNA [double-stranded DNA] with an appropriate translesion DNA synthesis (TLS) polymerase, followed by PCR amplification and next-generation sequencing (NGS) to generate genome-wide repair maps. We have termed this method translesion excision repair-sequencing (tXR-seq).”

With the new method, scientists can tag and collect the cast-off snippets of DNA that arise during DNA repair. The scientists sequence the snippets and then fit together the sequences to create a repair map of the genome.

Given the effort and expense required for DNA sequencing, the initial, proof-of-principle map published by Dr. Sancar and colleagues doesn't have the highest resolution possible. But it points the way toward the routine scientific use of such maps, especially as costs drop, to better understand how DNA-damaging events lead to disease and death.

This mapping technique should help answer several questions, such as:

• What dose of a toxin is needed to overwhelm the average person's nucleotide excision repair capacity?
• Which variations—and in which genes—give people more or less capacity to repair such DNA damage?
• Are there certain spots on the genome where successful repairs are inherently less likely?

Even with their initial, medium-resolution map, Dr. Sancar and colleagues were able to show that repairs of BPDE damage tend to occur more often when the BPDE-burdened guanine (G) is next to a cytosine (C) rather than a thymine (T) or adenine (A). This suggests there are “hotspots” of higher risk for BPDE-induced mutation.

“Understanding this bias in repair should help us better understand why exposures to toxins such as BaP tend to cause certain gene mutations,” noted Wentao Li, Ph.D., a postdoctoral researcher and lead author of the study.

In studies published in 2015 and 2016, Dr. Sancar and colleagues used earlier versions of their technique to map two other types of DNA-adduct damage: one wrought by UV light and the other by the common chemo drug cisplatin. Those mapping studies required an extra chemical step—removing the damage from an excised snippet before sequencing it—because the DNA-reading enzyme needed for the sequencing process would otherwise get stuck at the adduct. In contrast, the new technique employs “translesional” enzymes with dimensions that allow it to keep reading a strand of DNA, even when a bulky BPDE adduct is present.

“This new method can be applied to any type of DNA damage that involves nucleotide excision repair,” explained Dr. Sancar.

Drs. Sancar, Li, and their colleagues are now using the new technique to map DNA damage repair associated with other environmental toxins. Their next project focuses on aflatoxins, a family of mold-produced molecules often found in poorly stored nuts and grains. These toxins damage DNA and are major causes of liver cancer in developing countries.

The researchers are also performing more studies to uncover factors influencing where and whether nucleotide excision DNA repair occurs. To do that, they need to map sites of actual damage on the genome itself, not just the damaged snippets that are excised during repairs.

In one such project, they have developed a sensitive, high-resolution method for mapping actual DNA damage caused by UV light. By combining that method with repair mapping, they have found that the UV damage to DNA appears to be essentially uniform, although the repair process is not. Repair seems to be affected by a host of factors, including how actively a given stretch of DNA is being copied out to encode the making of proteins. They are currently applying this method to BaP to complement the repair map they have generated.

That again points to the likelihood of hotspots where repair is less likely to occur and mutations are more likely to arise.

“I'm certain,” said Dr. Sancar, “that all of this information will lead to a better understanding of why certain people are predisposed to cancer, and which smoking-related mutations lead to lung cancer specifically.”

And that, in turn, could have implications for the development of more targeting therapies down the line.