Stuart Linn

Formation, Prevention, and Repair of DNA Damage by Iron/Hydrogen Peroxide

Although oxygen is a powerful oxidant, the triplet ground state of dioxygen constitutes a kinetic barrier for oxidation of biological molecules, which are mostly singlet state (1). However, the unpaired orbitals of dioxygen can sequentially accommodate single electrons to yield O2 ., H2O2, the very reactive zOH, and water (Fig. 1, Reaction 1). The oxidative potential of atmospheric oxygen is maintained by the non-alignment of electron spins, and aerobic life is based upon harnessing energy via the catalytic spin pairing of triplet oxygen by the electron transport chain (2). The latter process occasionally errs, however, giving rise to O2 . and other reactive oxygen species (3) that cause cellular and genetic damage (4–7). Moreover, catabolic oxidases such as xanthine oxidase, anabolic processes such as nucleoside reduction, and defense processes such as phagocytosis also produce oxygen radicals. Although DNA is a biologically important target for reactive oxygen species, free O2 . is relatively unreactive with DNA (8). However, O2 . dismutates (via spontaneous or enzyme-catalyzed reactions) to produce H2O2 (Fig. 1, Reaction 2). O2 . can also reduce and liberate Fe from ferritin (9) (Fig. 1, Reaction 3) or liberate Fe from iron-sulfur clusters (10) (Fig. 1, Reaction 4); subsequently very reactive oxygen species can form via the Fenton reaction (Fig. 1, Reaction 5). Thus, the cytotoxic effects of O2 . (as well as of iron and H2O2) have been linked to DNA damage by way of the Fenton reaction (4, 11, 12) (Fig. 2).

Sequence-specific DNA Cleavage by Fe2+-mediated Fenton Reactions Has Possible Biological Implications

Preferential cleavage sites have been determined for Fe2+/H2O2-mediated oxidations of DNA. In 50 mm H2O2, preferential cleavages occurred at the nucleoside 5′ to each of the dG moieties in the sequence RGGG, a sequence found in a majority of telomere repeats. Within a plasmid containing a (TTAGGG)81human telomere insert, 7-fold more strand breakage occurred in the restriction fragment with the insert than in a similar-sized control fragment. This result implies that telomeric DNA could protect coding DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and aging. In micromolar H2O2, preferential cleavage occurred at the thymidine within the sequence RTGR, a sequence frequently found to be required in promoters for normal responses of many procaryotic and eucaryotic genes to iron or oxygen stress. Computer modeling of the interaction of Fe2+ with RTGR in B-DNA suggests that due to steric hindrance with the thymine methyl, Fe2+ associates in a specific manner with the thymine flipped out from the base stack so as to allow an octahedrally-oriented coordination of the Fe2+ with the three purine N7 residues. Fe2+-dependent changes in NMR spectra of duplex oligonucleotides containing ATGA versus those containing AUGA or A5mCGA were consistent with this model.

Oxidative Damage to DNA Constituents by Iron-mediated Fenton Reactions THE DEOXYGUANOSINE FAMILY

Damage by iron-mediated Fenton reactions under aerobic or anaerobic conditions to deoxycytidine, deoxycytidine-5′-monophosphate, d-CpC, d-CpCpC, and dCMP residues in DNA resulted in at least 26 distinguishable products. Of these, 24 were identified by high performance liquid chromatography retention times, radiolabeling, UV absorption spectra, chemical synthesis, fast atom bombardment mass spectrometry, high resolution fast atom bombardment mass spectrometry, and/or NMR. The nature of the products was qualitatively similar for each substrate except for d-CpC (and possibly d-CpCpC) under anaerobic conditions for which 5-hydroxy-deoxycytidine was uniquely present and 1-carbamoyl-1-carboxy-4-(2-deoxy-β—erythropentofuranosyl)glycinamide was uniquely absent. Damage to dC, d-CpC, and d-CpCpC but not to dCMP or DNA was largely quenched by ethanol, indicating that iron is strongly associated only with dCMP and DNA. The presence of oxygen had little effect with dC or dCMP but had quantitative and qualitative effects with d-CpC and a significantly quantitative but not a qualitative effect with DNA. NADH could drive the Fenton reaction to cause damage to the dC family in vitro, consistent with a previous proposal that NADH was the reducing agent for the Fenton reaction in vivo (Imlay, J.A., and Linn, S. (1988) Science 240, 1302-1309). Finally, the damage spectrum of the dC family by the Fenton reaction is compared with that by ionizing radiation and chemical mechanisms leading to the formation of the 24 identified products are proposed.

Mutations Specific to the Xeroderma Pigmentosum Group E Ddb− Phenotype

The activity of a damage-specific DNA-binding protein (DDB) is absent from a subset, Ddb−, of cell strains from patients with xeroderma pigmentosum group E (XP-E). DDB is a heterodimer of 127-kDa and 48-kDa subunits. We have now identified single-base mutations in the gene of the 48-kDa subunit in cells from the three known Ddb− individuals, but not in XP-E strains that have the activity. An A → G transition causes a K244E change in XP82TO and a G → A transition causes an R273H change in XP2RO and XP3RO. No mutations were found in the cDNA of the 127-kDa subunit. Overexpression of p48 in insect cells greatly increases DDB activity in the cells, especially if p127 is jointly overexpressed. These results demonstrate that p48 is required for DNA binding activity, but at the same time necessitate further definition of the genetic basis of XP group E.

The Naturally Occurring Mutants of DDB Are Impaired in Stimulating Nuclear Import of the p125 Subunit and E2F1-Activated Transcription

ABSTRACT The human UV-damaged-DNA binding protein DDB has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E), because a subset of XP-E patients lack the damaged-DNA binding function of DDB. Moreover, the microinjection of purified DDB complements the repair deficiency in XP-E cells lacking DDB. Two naturally occurring XP-E mutations of DDB, 82TO and 2RO, have been characterized. They have single amino acid substitutions (K244E and R273H) within the WD motif of the p48 subunit of DDB, and the mutated proteins lack the damaged-DNA binding activity. In this report, we describe a new function of the p48 subunit of DDB, which reveals additional defects in the function of the XP-E mutants. We show that when the subunits of DDB were expressed individually, p48 localized in the nucleus and p125 localized in the cytoplasm. The coexpression of p125 with p48 resulted in an increased accumulation of p125 in the nucleus, indicating that p48 plays a critical role in the nuclear localization of p125. The mutant forms of p48, 2RO and 82TO, are deficient in stimulating the nuclear accumulation of the p125 subunit of DDB. In addition, the mutant 2RO fails to form a stable complex with the p125 subunit of DDB. Our previous studies indicated that DDB can associate with the transcription factor E2F1 and can function as a transcriptional partner of E2F1. Here we show that the two mutants, while they associate with E2F1 as efficiently as wild-type p48, are severely impaired in stimulating E2F1-activated transcription. This is consistent with our observation that both subunits of DDB are required to stimulate E2F1-activated transcription. The results provide insights into the functions of the subunits of DDB and suggest a possible link between the role of DDB in E2F1-activated transcription and the repair deficiency disease XP-E.

DDB2 gene disruption leads to skin tumors and resistance to apoptosis after exposure to ultraviolet light but not a chemical carcinogen

Mutations in the human DDB2 gene give rise to xeroderma pigmentosum group E, a disease characterized by increased skin tumorigenesis in response to UV-irradiation. Cell strains derived from xeroderma pigmentosum group E individuals also have enhanced resistance to UV-irradiation due to decreased p53-mediated apoptosis. To further address the precise function(s) of DDB2 and the consequence of non-naturally occurring DDB2 mutations, we generated mice with a disruption of the gene. The mice exhibited significantly enhanced skin carcinogenesis in response to UV-irradiation, and cells from the DDB2–/– mice were abnormally resistant to killing by the radiation and had diminished UV-induced, p53-mediated apoptosis. Notably, the cancer-prone phenotype and the resistance to cellular killing were not observed after exposure to the chemical carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), to which mice carrying defective nucleotide excision repair genes respond with enhanced tumors and cell killing. Although cells from heterozygous DDB2+/– mice appeared normal, these mice had enhanced skin carcinogenesis after UV-irradiation, so that XP-E heterozygotes might be at risk for carcinogenesis. In sum, these results demonstrate that DDB2 is well conserved between humans and mice and functions as a tumor suppressor, at least in part, by controlling p53-mediated apoptosis after UV-irradiation.

Identification and Cloning of Two Histone Fold Motif-containing Subunits of HeLa DNA Polymerase ε

HeLa DNA polymerase ε (pol ε), possibly involved in both DNA replication and DNA repair, was previously isolated as a complex of a 261-kDa catalytic subunit and a tightly bound 59-kDa accessory protein. Saccharomyces cerevisiaepol ε, however, consists of four subunits: a 256-kDa catalytic subunit with 39% identity to HeLa pol ε p261, a 80-kDa subunit (DPB2) with 26% identity to HeLa pol ε p59, a 23-kDa subunit (DPB3), and a 22-kDa subunit (DPB4). We report here the identification and the cloning of two additional subunits of HeLa pol ε, p17, and p12. Both proteins contain histone fold motifs which are present also in S. cerevisiae DPB4 and DPB3. The histone fold motifs of p17 and DPB4 are related to that of subunit A of the CCAAT binding factor, whereas the histone fold motifs found in p12 and DPB3 are homologous to that in subunit C of CCAAT binding factor. p17 together with p12, but not p17 or p12 alone, interact with both p261 and p59 subunits of HeLa pol ε. The genes for p17 and p12 can be assigned to chromosome locations 9q33 and 2p12, respectively.

Impaired Regulation of Tumor Suppressor p53 Caused by Mutations in the Xeroderma Pigmentosum DDB2 Gene: Mutual Regulatory Interactions between p48DDB2 and p53

ABSTRACT Tumor suppressor p53 controls cell cycle progression and apoptosis following DNA damage, thus minimizing carcinogenesis. Mutations in the human DDB2 gene generate the E subgroup of xeroderma pigmentosum (XP-E). We report here that XP-E strains are defective in UV irradiation-induced apoptosis due to severely reduced basal and UV-induced p53 levels. These defects are restored by infection with a p53 cDNA expression construct or with a DDB2 expression construct if and only if it contains intron 4, which includes a nonmutated p53 consensus-binding site. We propose that both before and after UV irradiation, DDB2 directly regulates p53 levels, while DDB2 expression is itself regulated by p53.

Biochemical heterogeneity in xeroderma pigmentosum complementation group E

Cells from two patients with xeroderma pigmentosum complementation group E (XP-E) have been shown to lack an activity which binds specifically to UV-irradiated DNA (Chu and Chang, 1988). We investigated the occurrence of this binding activity in cell strains from nine additional, unrelated XP-E patients and found that all but one of these strains contained normal levels of the binding protein. Furthermore, the binding activity from these XP-E strains was indistinguishable from that of normal controls in thermal stability, behavior on ion-exchange chromatography, and electrophoretic mobility of protein-DNA complexes, indicating that there were no gross structural alterations in the protein. The association of XP-E with a deficiency in DNA-damage binding protein in cells from 3 of 12 XP-E patients (compared to 0 of 20 non-XP-E controls) is statistically significant (p less than 0.05), but there is no obvious correlation between the biochemical defect and the clinical or cellular characteristics of individual patients. Implications of these findings for the role of the binding protein in XP-E are discussed.

Electron microscopic studies of the mechanism of action of the restriction endonuclease of Escherichia coli B.

Reaction intermediates and products formed by the restriction endonuclease of Escherichia coli B with fd replicative form DNA substrates containing recognition sites in known positions and orientations have been characterized by electron microscopy. After exposure of these substrates to enzyme, loops of duplex DNA were frequently observed, usually at or near the termini. Analysis of the size and structure of the loops observed with various DNA substrates suggests that the enzyme binds initially to the recognition site then remains bound to the DNA in the region of this site while tracking towards a site of cleavage. Tracking appears to occur only on the 5′ side of the asymmetric recognition sequence, 5′ … T-G-A-(N)8-T-G-C-T … 3′; however, the location of the cleavage sites appears to be random, at least within certain limits of distance from the recognition site. Enzyme-DNA complexes remain intact even after the double-strand cleavage is completed, and this complex acts as a potent ATPase with no obvious function. This latter reaction might represent an artifactual uncoupling of ATP hydrolysis from the tracking of the enzyme along the DNA; alternatively, it might indicate an in vivo function for the enzyme of which we are unaware.