Radicals in Berkeley?
2015; Elsevier BV; Volume: 290; Issue: 14 Linguagem: Inglês
10.1074/jbc.x115.644989
ISSN1083-351X
Autores Tópico(s)Free Radicals and Antioxidants
ResumoIn a previous autobiographical sketch for DNA Repair (Linn, S. (2012) Life in the serendipitous lane: excitement and gratification in studying DNA repair. DNA Repair 11, 595–605), I wrote about my involvement in research on mechanisms of DNA repair. In this Reflections, I look back at how I became interested in free radical chemistry and biology and outline some of our bizarre (at the time) observations. Of course, these studies could never have succeeded without the exceptional aid of my mentors: my teachers; the undergraduate and graduate students, postdoctoral fellows, and senior lab visitors in my laboratory; and my faculty and staff colleagues here at Berkeley. I am so indebted to each and every one of these individuals for their efforts to overcome my ignorance and set me on the straight and narrow path to success in research. I regret that I cannot mention and thank each of these mentors individually. In a previous autobiographical sketch for DNA Repair (Linn, S. (2012) Life in the serendipitous lane: excitement and gratification in studying DNA repair. DNA Repair 11, 595–605), I wrote about my involvement in research on mechanisms of DNA repair. In this Reflections, I look back at how I became interested in free radical chemistry and biology and outline some of our bizarre (at the time) observations. Of course, these studies could never have succeeded without the exceptional aid of my mentors: my teachers; the undergraduate and graduate students, postdoctoral fellows, and senior lab visitors in my laboratory; and my faculty and staff colleagues here at Berkeley. I am so indebted to each and every one of these individuals for their efforts to overcome my ignorance and set me on the straight and narrow path to success in research. I regret that I cannot mention and thank each of these mentors individually. I was educated in public schools of the then-unincorporated West Hollywood district of Los Angeles. In high school, I was fortunate to have been exposed to “new math” and both classical and modern physics. In particular, Dr. Melvin Greenstadt was responsible for defining my enthusiasm for chemistry and science in general. This enthusiasm was not limited to me, as ten members of my 1958 high school graduating class applied to Caltech. Seven of us were accepted and enrolled into the class of 1962 with fewer than 200 members. The others were enticed by “new” physics and “new math” as majors, while I focused on chemistry and biology. How could I not pursue molecular biology/biochemistry, having taken courses and had numerous informal discussions at Caltech with the likes of Linus Pauling, Richard Feynman, George Beadle, Norman Horowitz, Norman Davidson, Renato Dulbecco, and Jerry Vinograd? Also, I did research in the laboratory of Henry Borsook, studying the expression of hemoglobin in his reticulocyte system, which went on to be used worldwide. Each one of these faculty members imparted knowledge of techniques and concepts that I applied in my later research. By chance, I also took an elective course on electroanalytical chemistry, which provided the necessary background for me to be able to study free radicals and reactive oxygen species (ROS) later in my career. With all of my exposure to biochemistry and molecular biology (and my California upbringing), I had only one obvious choice for graduate school: the newly formed biochemistry department at Stanford Medical School. For my thesis research, I worked with Bob Lehman on two DNases from Neurospora crassa, one of which was mitochondrial. In this way, not only did I learn how to manipulate DNA on gels, etc., but I also learned how to study mitochondria, an important aspect later in the study of free radicals. Moreover, I learned bacterial genetics from Dale Kaiser, Charles Yanofsky, and colleagues and aspects of DNA sequencing and oligonucleotide synthesis and manipulation from Gobind Khorana, who was on a sabbatical leave at Stanford while I was there. Finally, Joshua Lederberg, who was in an adjacent laboratory, made me appreciate the future applications of computer technology to biological studies, while Phil Hanawalt taught me about DNA repair. Although I did not work with Arthur Kornberg, we had regular graduate student seminars, during which he was extremely demanding; we all knew that if you could survive Arthur, you could survive giving a talk at any meeting anywhere. This pressure turned out to have one other important aspect: it prepared me for giving lectures to large undergraduate classes at Berkeley, in which hundreds of students consider it fair game to play “let's bait the prof.” Indeed, I am often asked why I seem so relaxed giving talks (or expert witness testimony), and my response is that “if I can survive a large Berkeley undergraduate class, I can be relaxed at any other presentation.” Between Caltech and Stanford, it would appear that I had been playing “let's meet who's who in biochemistry and molecular biology.” However, this did not include important personalities in Europe, so I went from Stanford to a postdoctoral position at the University of Geneva with Werner Arber. I studied host-controlled restriction and modification from a biochemical point of view and, in the process, learned more skills in bacterial genetics. Moreover, being in the department with Eduard Kellenberger, I learned techniques of electron microscopy, which were extremely useful in later studies of DNA damage and repair by ROS. I had one fortunate event during this period: I did a short study (supported by the European Molecular Biology Organization) with John Smith at the Medical Research Council in Cambridge, United Kingdom, to study the Escherichia coli B modification site. I could not have picked a luckier time for this visit. Of course, I interacted with and profited from discussions with the usual Medical Research Council Cambridge suspects, John Smith, Sydney Brenner, Fred Sanger, Francis Crick, etc., but also with a number of other visitors, including Joan and Tom Steitz (who were running DNA gels), Gobind Khorana (who was visiting), and Harry Noller. Given my exposure and interactions with so many pillars of modern biochemistry and molecular biology, I cannot believe what good fortune I have had in my life. Indeed, I am sure that in my case, whatever successes I have had in science were 95% due to luck in being at the right place with the right colleagues at the right time and 5% due to talent. In the fall of 1968, I arrived in Berkeley to take up a position of assistant professor of biochemistry. Having come from the calm, apolitical (at least on the surface) atmosphere of Switzerland, it was quite a shock to arrive in the midst of anti-war demonstrations, police barricades, and tear gas. However, with the realization that I had in my midst yet another collection of “pillars,” including Bruce and Giovanna Ames, Horace Barker, Melvin Calvin, John Clark, Harrison (Hatch) Echols, Seymour (Sy) Fogel, Dan Koshland, Bob Mortimer, Joe (Iron Man) Neilands, Howard Schachman, Wendell Stanley, Gunther Stent, Allan Wilson, and so many others, I began to study a number of problems, all related to DNA transactions, damage, and repair. I began my research with a continuation of studies of the restriction and modification enzymes of the EcoB system (1Lautenberger J.A. Linn S. The deoxyribonucleic acid modification and restriction enzymes of Escherichia coli B. I: Purification, subunit structure, and catalytic properties of the modification methylase.J. Biol. Chem. 1972; 247: 6176-6182Abstract Full Text PDF PubMed Google Scholar, 2Eskin B. Linn S. The deoxyribonucleic acid modification and restriction enzymes of Escherichia coli B. II: Purification, subunit structure, and catalytic properties of the restriction endonuclease.J. Biol. Chem. 1972; 247: 6183-6191Abstract Full Text PDF PubMed Google Scholar). Then, when John Clark informed us that phage restriction is defective in recB and recC mutants of E. coli, we characterized the RecBC(D) nuclease (3Karu A.E. MacKay V. Goldmark P.J. Linn S. The RecBC deoxyribonuclease of Escherichia coli K-12. Substrate specificity and reaction intermediates.J. Biol. Chem. 1973; 248: 4874-4884Abstract Full Text PDF PubMed Google Scholar, 4MacKay V. Linn S. The mechanism of degradation of duplex deoxyribonucleic acid by the RecBC enzyme of Escherichia coli K-12.J. Biol. Chem. 1974; 249: 4286-4294Abstract Full Text PDF PubMed Google Scholar). In 1974–1975, I took a sabbatical leave from Berkeley to study aging with Robin Holliday at the Medical Research Council in London. I was immediately impressed with the plethora of interests that Robin was pursuing with the utmost vigor. In addition to aging, he was studying recombination mechanisms in Ustilago maydis; DNA excision repair in Ustilago; and, what is truly impressive in retrospect, a role for what is now called “epigenetics” in development, differentiation, and stress responses (5Holliday R. Pugh J.E. DNA modification mechanisms and gene activity during development.Science. 1975; 187: 226-232Crossref PubMed Scopus (1424) Google Scholar). Indeed, coming into the lab every day was like reading an issue of Cell, in both the quality and variety of the science discussed. Although I did not find the fountain of youth in the Holliday lab, I did observe that in extracts from human cell strains, DNA polymerase fidelity dropped with passage number (6Linn S. Kairis M. Holliday R. Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.Proc. Natl. Acad. Sci. U.S.A. 1976; 73: 2818-2822Crossref PubMed Scopus (115) Google Scholar). After my return to Berkeley, I continued to study this phenomenon and finally concluded that what we were seeing was the replacement of the accurate, replicative polymerases with the less faithful, DNA repair “sloppier copier” lesion bypass enzymes (7Krauss S.W. Linn S. Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states.J. Cell. Physiol. 1986; 126: 99-106Crossref PubMed Scopus (23) Google Scholar). However, in the process, we also discovered a new replication DNA polymerase, polymerase ∈, which we went on to characterize (8Syväoja J. Suomensaari S. Nishida C. Goldsmith J.S. Chui G.S. Jain S. Linn S. DNA polymerases α, δ and ∈: three distinct enzymes from HeLa cells.Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 6664-6668Crossref PubMed Scopus (152) Google Scholar). During the winter just prior to my year in the Holliday lab, my family went skiing at Squaw Valley. Unfortunately, it rained continually while we were there, and my two preschool children were suffering extreme bouts of cabin fever. Fortunately for me, however, the cabin that we rented was only a few hundred meters from the site of the ICN-UCLA (now Keystone) meeting on DNA repair. Escaping from preschool chaos to the meeting, I quickly concluded that there was really very little true understanding of the chemistry and enzymology of DNA damage and repair in mammalian cells and vowed to enter that field when I returned from my sabbatical leave. Conversely, I also realized that quite a bit was known about the E. coli enzymes that took part in DNA repair, so these could be relatively easily purified and utilized as reagents for studying the mammalian systems. We studied activities that recognized and cleaved DNA at baseless (apurinic or apyrimidinic (AP)) sites and found two classes. Class II (now called Apn1, ADE/Ref-1, or HAP in various laboratories) cleaves DNA to leave a 3′-hydroxyl group and a 5′-baseless sugar phosphate. A property of HAP that we discovered is that it is inhibited by NAD+ and no other pyridine nucleotide, and it is also inhibited by adenine. We proposed that the cell avoids extensive base excision repair when it has reduced energy stores. Perhaps related is the fact that sirtuins are active only in the presence of stores of NAD+. The second group, which we called Class I, cleaves damaged DNA such that the final product contains a baseless fragment on the 3′ terminus. This group of enzymes was ultimately found to be a class that also contained DNA glycosylase activity for the damages; it could cleave off the damaged base prior to cleaving the phosphodiester bond. Moreover, we noted that this group of enzymes tended to recognize damages that could be caused by ROS. At least some members of this enzyme class were also present in mitochondria. Reference 9Linn S. Life in the serendipitous lane: excitement and gratification in studying DNA repair.DNA Repair. 2012; 11: 595-605Crossref PubMed Scopus (1) Google Scholar gives a more detailed summary of this work, along with another major study in our laboratory: the mammalian DNA damage-binding protein (DDB) and its role in p53 responses and preventing various degenerative diseases of aging. The topic of study in our laboratory that produced the most unanticipated and novel results was the follow-up on our observations that so much of a prokaryotic or eukaryotic cell's capacity for DNA damage recognition and repair was directed toward damages by ROS. Because it would have been difficult to irradiate large volumes of bacterial or mammalian cell cultures with γ-rays or ultraviolet light, we decided to use hydrogen peroxide as the damaging agent. Initial attempts to induce enzymes in E. coli that might repair oxidative damage were carried out by Bruce Demple, a graduate student in the laboratory at the time who went on to do postdoctoral work in the laboratory of Tomas Lindahl, and an undergraduate, James Halbrook, an undergraduate researcher who went on to graduate school at UCLA. These experiments initially appeared not to have been successful. However, on reanalyzing the results after they left Berkeley, Demple and Halbrook realized that the treated cells became resistant to subsequent hydrogen peroxide exposure. Lindahl very generously allowed Demple to explore the observation further in his laboratory, and Demple reproduced the results and also showed that the treatment made cells resistant to γ-radiation as well. Demple and Halbrook published their results in Nature (10Demple B. Halbrook J. Inducible repair of oxidative DNA damage in Escherichia coli.Nature. 1983; 304: 466-468Crossref PubMed Scopus (289) Google Scholar). Subsequently, back at Berkeley, a graduate student in my laboratory, James Imlay, wanted to study the chemistry/biochemistry of the adaptation. (Simultaneously, Mike Christman, Gisela Storz, Louis Tartaglia, and others in Bruce Ames' laboratory went on to study the molecular biology of the induced adaptation.) Fortunately, Imlay took quite seriously my mantras of “believe your results” and “if you believe you are right, fight for your beliefs regardless of who disagrees.” Imlay began his studies with the obvious necessity of knowing the toxicity of H2O2 exposure for E. coli. He shortly came to me with a curve for the wild-type strain AB1157 (Fig. 1). He was excited to point out that there was a dip and recovery near 1–2 mm in the shoulder region of the survival curve for AB1157 upon challenge with H2O2, after which the shoulder continued until ∼15–20 mm. Looking at the curve, I immediately responded that the dip was not going to be reproducible, that it was most likely due to a variety of factors, and that we ought to move on with our studies. Fortunately, Imlay ignored my remarks, went back to the lab, and several weeks later, showed me how wrong I had been by producing an effect with orders of magnitude differences when using strains lacking exonuclease III (defective in excision repair), active RecA protein (defective in recombination), or active DNA polymerase I (defective in both) (Fig. 1). He quickly went on to show that the effect was exaggerated during anoxic growth and that it required active metabolism (i.e. a carbon source), but not protein synthesis (i.e. it was not affected by chloramphenicol). When we submitted these results to the Journal of Bacteriology for publication, a reviewer stated, “This is the most obscure and unintelligible paper which I have ever been asked to review.” We took the statement to imply both that the results were novel and exciting and that Imlay's undergraduate double major in English was not quite beneficial for writing a scientific paper. In any event, the paper was eventually accepted (11Imlay J.A. Linn S. A bimodal pattern of killing of DNA repair-defective or anoxically grown Escherichia coli by hydrogen peroxide.J. Bacteriol. 1986; 166: 519-527Crossref PubMed Scopus (266) Google Scholar). Looking back at the enhanced sensitivity of E. coli to H2O2 when grown anaerobically, I propose that one ought to study the organism in anaerobic and aerobic environments. For example, many DNA repair mutant combinations are toxic under aerobic (but not anaerobic) conditions, and the DNA polymerase polA1 mutant is very “sick” in aerobic conditions, but normal when grown anaerobically. Moreover, when studying E. coli as a contributor to the intestinal microbiota, in vitro experiments should certainly be done anaerobically. While continuing his studies in our laboratory (12Linn S. Imlay J.A. Toxicity, mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide.J. Cell Sci. Suppl. 1987; 6: 289-301Crossref PubMed Google Scholar13Imlay J.A. Linn S. Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide.J. Bacteriol. 1987; 169: 2967-2976Crossref PubMed Scopus (302) Google Scholar, 14Imlay J.A. Chin S.M. Linn S. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro.Science. 1988; 240: 640-642Crossref PubMed Scopus (1278) Google Scholar15Imlay J.A. Linn S. DNA damage and oxygen radical toxicity.Science. 1988; 240: 1302-1309Crossref PubMed Scopus (1688) Google Scholar), Imlay went on to show that similarly complex response curves to peroxide exposure are obtained when monitoring mutagenesis, induction of phage λ, or cell division delay. He defined the two sections of the curve as Mode I (that seen at lower peroxide concentrations, with the dip and rise) and Mode II (that seen at higher millimolar peroxide concentrations, which was independent of peroxide concentration). Realizing that NADH accumulates in anaerobic cells and in those with blocked metabolism, he tested the effect of exposing ndh (NADH dehydrogenase) mutants to H2O2 and found that they also gave complex curves analogous to those obtained with anaerobic cells or cells treated with KCN. Imlay proposed an explanation for the Mode I response (Fig. 2): glucose provides electrons for NADH production. NADH normally provides four electrons for the reduction of oxygen to water or two electrons for the reduction of peroxides to water. However, on occasion, NADH can provide a single electron for the reduction of H2O2 and form the extremely potent hydroxyl radical. However, H2O2 can also oxidize the hydroxyl radical to the much less potent superoxide radical via the Haber-Weiss reaction. H2O2+HO˙→O2√¯+H2O+H+The latter would normally be relatively easily removed by superoxide dismutase. The Haber-Weiss reaction would explain the rise and subsequent drop in H2O2 toxicity, as schematized in Fig. 2. What could be the mediator of the one-electron transfer from NADH to hydrogen peroxide? Imlay proposed that it was iron (14Imlay J.A. Chin S.M. Linn S. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro.Science. 1988; 240: 640-642Crossref PubMed Scopus (1278) Google Scholar, 15Imlay J.A. Linn S. DNA damage and oxygen radical toxicity.Science. 1988; 240: 1302-1309Crossref PubMed Scopus (1688) Google Scholar). NADH would reduce ferric ion to ferrous ion, which could then generate a hydroxyl radical via the Fenton reaction. Fe2++H2O2+H+→Fe3++HO˙+H2OImlay then went on to extend these studies as a postdoctoral fellow in Irwin Fridovich's laboratory and afterward as an independent investigator in his own laboratory. Our laboratory went on to study the chemistry of DNA damage by the Fenton reaction with ferrous ion or by the related reaction with NADH and ferric ion. In the former case (16Luo Y. Han Z. Chin S.M. Linn S. Three chemically distinct types of oxidants formed by iron-mediated Fenton reactions in the presence of DNA.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 12438-12442Crossref PubMed Scopus (132) Google Scholar, 17Henle E.S. Linn S. Formation, prevention and repair of DNA damage by iron/hydrogen peroxide.J. Biol. Chem. 1997; 272: 19095-19098Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar18Henle E.S. Han Z. Tang N. Rai P. Luo Y. Linn S. Sequence-specific DNA cleavage by Fe2+-mediated Fenton reactions has possible biological implications.J. Biol. Chem. 1999; 274: 962-971Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), we were literally thrilled to generate a DNA-nicking response in vitro that was analogous to the Mode I/Mode II-shaped curve that we had observed in vivo in E. coli (Fig. 3). In the presence of ferrous iron, DNA nicking was maximal in the micromolar range of hydrogen peroxide; became quenched in the low millimolar range; and was then relatively independent of peroxide concentration up to ∼10 mm, after which it increased somewhat. When ethanol, a hydroxyl radical scavenger, was added at various concentrations, it eliminated the Mode I (but not Mode II) portion of the curve. Above 10 mm, ethanol eliminated the slight rise in nicking seen at high concentrations of H2O2. From these results, we hypothesized that there were three types of Fenton reaction radicals that can cause DNA nicking. Type I radicals, which cause Mode I damages, are formed by ferrous ions that are loosely coordinated with DNA and thus moderately resistant to ethanol and sensitive to H2O2 oxidation. Type II radicals, which cause Mode II damages, are tightly base-coordinated and thus very resistant to ethanol and H2O2 oxidation. Finally, Type III radicals are formed by ferrous ions that are free in solution and thus very sensitive to ethanol and sensitive to H2O2 oxidation. As was appreciated later, radicals formed by peroxide reacting with ferrous ions sequestered by molecules such as DNA are quite reactive, but they are not chemically identical to those formed with free ferrous ions in solution. This type of radical is now denoted as a ferryl radical. Although the superoxide anion is not particularly reactive with purified DNA in vitro, it is highly mutagenic and DNA damaging in vivo (17Henle E.S. Linn S. Formation, prevention and repair of DNA damage by iron/hydrogen peroxide.J. Biol. Chem. 1997; 272: 19095-19098Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). It undergoes dismutation either spontaneously or catalyzed by superoxide dismutase to form hydrogen peroxide. 2O2˙¯+2H+→O2+H2O2Moreover, it can reduce and liberate ferric ion from ferritin or ferrous ion from iron-sulfur clusters, thus allowing the generation of very reactive oxygen species, such as hydroxyl radical, via Fenton or related reactions. In a subsequent study in our laboratory at Berkeley, Ernst Henle, Priyamvada Rai, Yongzhang Luo, Zhengxu Han, and Ning Tang explored which DNA base sequences are cleaved by ferrous ion and H2O2 in vitro (18Henle E.S. Han Z. Tang N. Rai P. Luo Y. Linn S. Sequence-specific DNA cleavage by Fe2+-mediated Fenton reactions has possible biological implications.J. Biol. Chem. 1999; 274: 962-971Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). We were astounded to find that the specificities were virtually as great as those seen with restriction enzymes. Type I oxidants (formed in 0.5 mm H2O2) cleaved DNA at the sequence RT↓GR (where R = A or G, and ↓ indicates the location of the cleavage.) On the other hand, Type II oxidants (formed in 50 mm H2O2) cleaved DNA at R↓G↓G↓G or weakly at T↓G↓G↓G. Cleavages occurred 5′ to each of the dG residues, but with decreasing frequency going from 5′ to 3′. RGGG is contained in the majority of telomere repeats. In the same study, when we exposed a related plasmid containing a human telomere to Type II oxidants, the telomere was cleaved with the same frequency at each of the repeats as that of individual RGGG sequences elsewhere in the plasmid. Evidently, telomeres can “soak up” free iron, perhaps linking iron load, telomere damage, and shortening to aging phenomena. In a subsequent collaboration with David Wemmer in the chemistry department (19Rai P. Wemmer D.E. Linn S. Preferential binding and structural distortion by Fe2+ at RGGG-containing DNA sequences correlates with enhanced oxidative cleavage at such sequences.Nucleic Acids Res. 2005; 33: 497-510Crossref PubMed Scopus (33) Google Scholar), Rai used 1H NMR to characterize the binding of Fe2+ to the duplex oligonucleotide CGAGTTAGGGTAGC/GCTAACCCTAACTCG and 7-deazaguanine variants of it. She showed that Fe2+ binds preferentially to the GGG sequence, most strongly toward its 5′-end. Moreover, she showed that binding involves two adjacent guanine N7 moieties and that it is accompanied by large changes in specific imino, aromatic, and methyl proton chemical shifts such that a distorted structure forms at the binding site and also 2 bp 3′ to the GGG sequence. Thus, the zero-order dose response seen under Mode II conditions is apparently due to the rate-limiting step of reorganizing the distorted structure to expose the bound Fe2+ to H2O2 to enable formation of a DNA-damaging ferryl radical. Looking at the RTGR sequence, Henle et al. (18Henle E.S. Han Z. Tang N. Rai P. Luo Y. Linn S. Sequence-specific DNA cleavage by Fe2+-mediated Fenton reactions has possible biological implications.J. Biol. Chem. 1999; 274: 962-971Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar) noted that it is biologically important. It is contained in the (ATGGA)n centromeric repeats and frequently found to be required in promoters for normal responses of many prokaryotic and eukaryotic genes to iron or oxygen stress. Perhaps most dramatic is the case of the human AP endonuclease promoter, which contains three required sites in palindromic variants of RTGR for binding of the human upstream factor for its regulation. Henle et al. (18Henle E.S. Han Z. Tang N. Rai P. Luo Y. Linn S. Sequence-specific DNA cleavage by Fe2+-mediated Fenton reactions has possible biological implications.J. Biol. Chem. 1999; 274: 962-971Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar) then modeled the interaction of Fe2+ with RTGR in B DNA. The modeling predicted that the thymine methyl group provides steric hindrance to binding such that the thymine must flip out of the helix to allow coordination of the Fe2+ with the three purine N7 residues. Preliminary NMR studies were consistent with such a model. Rai et al. (20Rai P. Cole T.D. Wemmer D.E. Linn S. Localization of Fe2+ at an RTGR sequence within a DNA duplex explains preferential cleavage by Fe2+ and H2O2.J. Mol. Biol. 2001; 312: 1089-1101Crossref PubMed Scopus (61) Google Scholar) then collaborated once again with David Wemmer to study the binding by 1H NMR. They showed that Fe2+, but not Fe3+, preferentially binds to the RTGR sequence because of its unique structure, but the binding is relatively weak and reversible and does not strongly perturb the structure in duplex DNA. Because the binding is weak, the Fe2+ is subject to oxidation in the unbound state by H2O2. The resulting HO• is quenched in a first-order Haber-Weiss reaction. These results would explain the peculiar dose response for Mode I phenomena and led our laboratory to propose that the presence of RTGR in promoters of genes regulating responses to stress by ROS allows the promoters to detect the oxidation state of free iron cations in the cell and respond by positively or negatively regulating these genes. For example, the strong binding of Fe2+ by the human AP endonuclease promoter would act to regulate AP endonuclease production in anticipation of DNA damage by Fenton-type reactions due to the presence of free Fe2+. A final subject of our ROS studies had to do with the role of nicotinamide nucleotides in the phenomena (21Brumaghim J.L. Li Y. Henle E. Linn S. Effects of hydrogen peroxide upon nicotinamide nucleotide metabolism in Escherichia coli. Changes in enzyme levels and nicotinamide nucleotide pools and studies of the oxidation of NAD(P)H by Fe(III).J. Biol. Chem. 2003; 278: 42495-42504Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), a forerunner to conclusions in our laboratory and many others on the importance of this group of molecules in regulating cell metabolism and responses to stress. Following his initial observation that ndh mutants of E. coli were especially sensitive to killing by H2O2, Imlay had shown that NADH could be oxidized by peroxide in the presence of catalytic amounts of Fe3+, and, more to the point, DNA could be damaged by peroxide and NADH in the presence of catalytic amounts of Fe3+ (15Imlay J.A. Linn S. DNA damage and oxygen radical toxicity.Science. 1988; 240: 1302-1309Crossref PubMed Scopus (1688) Google Scholar). These observations were then reproduced (Fig. 4A) and extended (21Brumaghim J.L. Li Y. Henle E. Linn S. Effects of hydrogen peroxide upon nicotinamide nucleotide metabolism in Escherichia coli. Changes in enzyme levels and nicotinamide nucleotide pools and studies of the oxidation of NAD(P)H by Fe(III).J. Biol. Chem. 2003; 278: 42495-42504Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) in our laboratory by Julia Brumaghim, Ying Li, and Henle, who observed that although NADH could drive DNA nicking in the system, NADPH could not. Moreover, NADPH inhibited the nicking driven by NADH (Fig. 4B). The interactions of NADH and NADPH with Fe3+ and Ga3+ were also studied by 1H, 13C, and 31P NMR spectroscopy (21Brumaghim J.L. Li Y. Henle E. Linn S. Effects of
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