SCIENCE & TECHNOLOGY
DOING DNA DAMAGE ASSESSMENT The best studied DNA lesion arising from oxidants' attack may not be the only toxic one AMANDA YARNELL, C&EN WASHINGTON
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HE OXIDIZED DNA BASE 8-OX-
oguanine is widely considered public enemy number one among the kinds of DNA damage caused by exposure to radiation or reactive oxygen species. Remarkably, this modified guanine— which can giveriseto dangerous mistakes in the genetic code—differs from its undamaged parent by only one atom. The ease with which this lesion is formed and detected has made 8-oxoguanine the subject of intense research. That, coupled with a number of other factors, has made it one of the best characterized DNA lesions, notes Sheila S. David, an associate chemistry professor at the University of Utah. But recent work suggests that other DNA modifications caused by oxidation may also endanger cells. Cells hold dear the genetic information contained in their DNA and work diligently to maintain its sequence. The threat of DNA damage comes in manyflavors.Among the troublemakers are breaks in the DNAhelix and modification of DNA bases by anticancer drugs, alkylating agents, or oxidants. One of the best studied of these modifications, oxidative damage, is caused by reactive oxygen species (most often hydroxyl radicals) as well as other chemical agents. Produced by everything from the sun's ultraviolet light to chemicals in our diet to normal metabolic activity, these reactive species modify the nucleotide bases that make up DNA. One of the most common alterations is the addition of a ketone group to a guanine base to form the aforementioned 8-oxoguanine—officially known as 8-oxo-7,8-dihydroguanine.
pair where a guanineicytosine base pair should be. Suchguanine:cytosine to thymineiadenine mutations can be devastating, David points out, because cells rely on the structural integrity of their DNA to transmit genetic information from one generation to the next. In response, cells have developed a comprehensive DNArepair system to find and remove 8-oxoguanine—a fact that she says further illustrates this lesion's importance.
THIS SYSTEM begins with prevention: An enzyme called Mthl cleanses the pool of nucleotide triphosphate DNA-building blocks of 8-oxoguanine lesions. This stymies the incorporation ofpreformed 8oxoguanine into DNA during replication. To take care of 8-oxoguanine lesions that have already made their way into the DNA duplex, cells turn to a DNA repair enzyme called oxoguanine glycosylase/lyase (Oggl). Ogglfindsand removes any 8-oxoguanines that are opposite cytosine in duplex DNA. Other enzymesfillin the gap, restoring the undamaged guanineicytosine pair. But on occasion, damaged DNAstrands are accidentally replicated before an 8-oxoguanine lesion is removed, creating an 8-oxoguanine: adenine mismatch. In these cases, an enzyme called Myh catalyzes the removal of the unwanted adenine. Other enzymes then replace it with cytosine, yielding an 8-oxoguanine:cytosine base pair that is repaired via the Oggl pathway Together, these factors—the SEARCH AND DESTROY The repair enzyme 0gg1 (gray) recognizes 8-oxoguanine frequency with which this lesion forms, its tendency to cause DNA (blue) by extruding the modified base into its active site. The estranged cytosine is shown in mutations, and the complex DNA repair machinery cells have red; the DNA helix is colored gold. evolved to combat it—have made 8-oxoguanine pairs up with adenine (nor- 8-oxoguanine the center of attention. However, as recent research is emphamally thymine's partner in the DNA helix) instead of cytosine (guanine's normal sizing, 8-oxoguanine is not the only player. More than 50 lesions that arisefromthe partner). So when cells replicate their DNA, attack of oxygen radicals on DNA have some DNA polymerases tend to mistak- been identified so far. 'And there's no way enly pair 8-oxoguanine with adenine of estimating how many we haven't found instead of cytosine. If this 8-oxo- yet," notes Mirai Dizdaroglu, a chemist at guanineiadenine mismatch isn't repaired the National Institute of Standards & before the cell again replicates its DNA, Technology Dizdaroglu has shown that hydroxy! radit will give rise to a thymineiadenine base The interest in this alteration was natural. For one, 8-oxoguanine lesions give rise to potentially deleterious DNA mutations. The extra oxygen atom on 8-oxoguanine allows the damaged base to adopt a glycosidic bond conformation that is different from the one favored by guanine bases in the DNA helix. When it assumes this abnormal conformation,
"It's important to remember that the most prevalent DNA lesion is not necessarily the most dangerous one to the cell.
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SCIENCE & TECHNOLOGY icals react with all four DNA bases, both The stumbling block for 8-oxoguanine Cadet and his coworkers have also tried in vitro and in vivo, spawning a plethora of to determine the abundance ofvarious ox- was removed by Francis Johnson, a proproducts. He and his coworkers at NIST idative lesions formed in leukemia cells ex- fessor of chemistry and pharmacological are using gas chromatography/mass spec- posed to 7-radiation. They found that near- sciences at the State University of New trometry and liquid chromatography/mass ly three times as much Fapy-G as %rk, Stony Brook, who has carvedacareer spectrometry to characterize this soup of 8-oxoguanine is formed under lesions. The lesions theyVe found to com- these conditions [Radiât Res., monly form in vitro include 8-oxoguanine, 157,589 (2002)}. Others, in8-oxoadenine, 2,6-diamino-4-hydroxy-5- cluding Dizdaroglu, have found formamidopyrimidine (Fapy-G), 4,6-di- similar ratios ofFapy-G to 8-oxamino-5-fonnamidopyrimidine (Fapy-A), oguanine in vivo. 5-hydroxycytosine, and 5-hydroxyuracil. "It's important to remember, But the relative yields of these lesions however, that the most prevadepend on experimental conditions, he lent DNA lesion is not necescautions. Because there's less oxygen in the sarily the most dangerous one to the cell," out of making oligonucleotides contain nucleus than elsewhere in the cell, Diz- Cadet is quick to point out. ing defined DNA lesions. He worked out daroglu suspects that the soup of lesions To probe the danger of individual DNA a synthetic strategy for 8-oxoguanine-conformed with hydroxyl radicals under low lesions to cells, chemists have had to figure taining oligonucleotides in the early '90s oxygen conditions—which is dominated out a way to synthesize DNA containing [Cbem. Res. Toxicol. 5,608 (1992)}. by 8-oxoguanine and FapyThe availability of syn G—may paint the most acthetic stretches ofDNAconDAMAGED GOODS curate picture of the oxidataining single 8-oxoguanine Oxidation of guanine gives rise to 8-oxoguanine as well as tive damage that cells have lesions "changed everything,'' a number of other potentially dangerous DNA lesions to cope with. Verdine tells C&EN. It al lowed chemists to probe how the lesion affects the struc APART FROM hunches, HN ture of the surrounding however, it's hard to tell Y^NH DNA. It made it possible to whether the spectrum and NH9 measure just how mutagenic abundance oflesions formed this particular lesion is. And in these in vitro experiments it permitted Verdine and oth accurately reflect what's goers to study how repair pro ing on in the cell. Of the 50teins interact with the lesion. plus lesions identified in in Verdine's lab is usingX-ray vitro experiments, only a 8-Oxoguanin· crystallography to determine dozen or so have been dehow Oggl hunts for and re tected in vivo, according to pairs 8-oxoguanine lesions in Jean Cadet, scientific adviser a vast sea of undamaged at Commissariat à l'Energie DNA. Their structure of Atomique (CEA), Grenoble, Note: Bases are Oggl bound to an oligonu France. Cadet has been tryshown schematically, cleotide containing 8-ox ing to measure the cellular as they would be oguanine shows that the levels of various oxidative attached to the DNA backbone. damaged base is thrust out DNA lesions for more than of the DNA helix and into a decade. Oggl's active site [Nature, And this is no easy matter, Ouanint 403, 859 (2000)1. "Amaz he points out. A decade ago, ingly, Oggl doesn't interact scientists thought that tens H with 8-oxoguanine's most ofthousands of8-oxoguanine η N ^ O NH distinctive feature, its extra lesions were present in each oxygen atom," Verdine says. cell's copy of the genome at Instead, he says, the enzyme any given time, Cadet tells differentiates between gua C&EN. But in part by maknine and 8-oxoguanine by ing the switch from detectway of a single hydrogen ing 8-oxoguanine electrobond to the damaged base's chemically to detecting it by Spiroiminohydantoin Guanidlnohydantoin N7 proton. more sensitive and spécifie But because the critical mass spectrometric methods, his lab has shown that this number is actu- specific, often chemically unstable modi N7 proton is tucked out of reach in the ally on the order of a few hundred 8-ox- fications. "In the wholefieldofDNA dam DNA helix, Verdine and others have re oguanines.This translates into only a few 8- age, the chemical synthesis of lesions is a mained puzzled about how Oggl hunts for oxoguanines per 10 million normal bases. major stumbling block," notes Gregory L. 8-oxoguanine lesions. Some have suggest But even this amended number remains Verdine, a professor of chemical biology ed that the enzyme may flip each and every at Harvard University base out of the helix in its search for 8-oxcontroversial.
The availability of synthetic stretches of DNA containing single 8-oxoguanine lesions "changed everything/'
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oguanine lesions. Others have proposed that Oggl might probe the helix in other, less drastic ways. Oggl's search mechanism remains mysterious, but how it removes 8-oxoguanine is better understood. Like other DNAglycosylases, Oggl catalyzes removal of the damaged base via a transient Schiff base intermediate. Recently, \ferdine has shown that Oggl uses this strategy to clip 8-oxoguanine from the DNA backbone. The enzyme uses the 8-oxoguanine it produces as a cofactor in a subsequent catalytic step that severs the backbone [Nat Struct. Biol, 10, 204 (2003); C&EN, Feb. 24, page 8}. In hopes of gathering this kind of detail about the cellular processing and biological relevancy of other kinds of oxidative DNA modifications, chemists are racing to make oligonucleotides containing these oxidation lesions. Marc M. Greenberg, an organic chemistry professor at Johns Hopkins University, recently developed a method for constructing oligonucleotides containing defined FapyG lesions \J. Am. Chem. Soc, 124, IMPERFECT PAIR In the DNA helix, guanine (above left) normally pairs up with 3263(2002)}. cytosine (above right.) But when guanine is oxidized to 8-oxoguanine (below left; It's been shown that Fapy-G le- oxidized carbon is starred ), the damaged base can alter its glycosidic bond sions form more readily than 8-ox- conformation and mispair with adenine (below right). This mispairing can lead to oguanine under some conditions, dangerous DNA mutations. Hydrogen bonds are shown as dashed white lines. (Green, both in vivo and in vitro. But until carbon; blue, nitrogen; red, oxygen; gray, hydrogen.) very recently, Fapy-G lesions have been underappreciated—in part because mann and Steven R. Tannenbaum have in- lian R. Sampson of the University of Wales oligonucleotides containing Fapy-G were vestigated whether certain higher oxida- College of Medicine have shown that synthetically inaccessible, Greenberg says. tion products of 8-oxoguanine can cause there's a direct link between inherited deHis lab has shown that Fapy-G can cause DNA mutations in bacteria [Biochemistry, fects in Myh—one of the enzymes that the same deleterious guanine:cytosine to 41,914 (2002)}.They found that the more helps repair 8-oxoguanine lesions—and thymine:adenine mutations in vitro as 8- highly oxidized products are considerably patients' propensity for contracting colon oxoguanine does [J. Am. Chem. Soc, 124, more mutagenic than 8-oxoguanine. cancer [Nat. Genet, 30,227 (2002). They 7278 (2002)]. Plus, he tells C&EN, his lab Burrows has figured out how to syn- found that members of a British family afhas unpublished evidence showing that thesize oligonucleotides containing two fected by multiple colorectal cancers have Fapy-G causes mutations in bacteria at a other products of further oxidation of 8- two different mutations in the gene codsignificant rate. oxoguanine: spiroiminodihydantoin and ing for Myh. These two mutant forms of Could FapyG be more dangerous than guanidinohydantoin. She and David have Myh both remove adenine mispaired with 8-oxoguanine? "It's too early to tell," shown that these lesions are poorly re- 8-oxoguanine much less efficiently than Greenberg says. "But it shouldn't be over- paired and that polymerases tend to pair the wild-type enzyme does. The reduced activity ofthe mutant Myh looked." them with adenine or guanine—but nevNor should we overlook the products er guanine's normal partner, cytosine. Bur- enzymes found allows mutations to perof further oxidation of 8-oxoguanine, says rows and Essigmann are now collaborating sist, permitting mutations to accumulate organic chemist Cynthia J. Burrows, pro- to measure how mutagenic these two le- in a particular gene that causes colon cancer in these patients. 'This is thefirstevifessor of chemistry at the University of sions are to bacteria. dence for an association between inheritUtah. 8-Oxoguanine's very low redox potential makes it highly susceptible to fur- WHICHEVER LESION proves to be the ed defects in the repair ofoxidative lesions ther oxidation. Burrows suspects these fur- most toxic DNAmodification arising from and predisposition for cancer in humans," ther oxidized products will prove even oxidative damage, new work linking ox- David tells C&EN. 'And it's sure to spark more dangerous than 8-oxoguanine. idative DNA damage to cancer is sure to even more interest in the chemistry of these lesions and the biochemistry of their In fact, Massachusetts Institute of Tech- continue to drive interest in this area. nology chemistry professors John EssigUtah's David and medical geneticistJu- repair," she adds. • HTTP://WWW.CEN-ONLINE.ORG
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