Action of gamma endonuclease on clustered lesions in irradiated DNA

Summary Irradiation of DNA in situ i.e. in phage particles or in the cell leads to alterations of single DNA nucleotides as well as to clustered lesions such as double strand breaks or unpaired DNA regions the latter being sensitive to digestion by S 1 nuclease. A contribution will be made to the configuration of such S 1-nuclease-sensitive sites (S 1 sites). DNA from irradiated lambda phage containing S 1 sites was treated with gamma endonuclease fromM. luteus which is known to split the nucleotide strand at the position of oxidized pyrimidine base. It was found that the gamma endonuclease induces double-strand breaks at some of the S 1 sites indicating double base damage within this site. However, half of the S 1 sites are not converted into a double-strand break by the gamma endonuclease, indicating base damage only on one strand within the unpaired region.Dedicated to Prof. W. Jacobi on the occasion of his 60th birthday     

Double lesions are produced in DNA oligomer by ionizing radiation and by metal-catalyzed H2O2 reactions

It was demonstrated previously that double lesions are produced in DNA by ionizing radiation. These double lesions consist of adjacent nucleotides each bearing a modified base. The goal of the present investigation was to determine whether Fenton chemistry can generate the same kind of lesions. DNA oligomers were exposed to metal-catalyzed H(2)O(2) reactions, and the products were characterized by chromatography and by mass spectrometry. Double lesions are produced by this treatment in which deoxyguanosine is oxidized to 8-oxo-7,8-dihydrodeoxyguanosine and an adjacent pyrimidine nucleoside is degraded to a formamido remnant.     

Delayed repair of radiation induced clustered DNA damage: friend or foe?

A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a "friend", leading to cell killing in tumour cells or as a "foe", resulting in the formation of mutations and genetic instability in normal tissue.     

Double base lesions in DNA X-irradiated in the presence or absence of oxygen

Previously, double lesions in which two adjacent bases are modified were identified in DNA oligomers exposed in solution to ionizing radiation. However, the formation of such lesions in polymer DNA had not been demonstrated. Using reference oligomer containing a specific double lesion and employing liquid chromatography-mass spectrometry (LC-MS), it was possible to show directly that double lesions are formed in irradiated calf thymus DNA. The double lesion in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized to 8-oxoguanine was detected in DNA X-irradiated in oxygenated aqueous solution. The double lesion in which the methyl carbon atom of a thymine base is covalently linked to carbon at the 8-position of an adjacent guanine base was detected in DNA irradiated in a deoxygenated environment.     

Structure of a photoactive rhodium complex intercalated into DNA

Intercalating complexes of rhodium(III) are strong photo-oxidants that promote DNA strand cleavage or electron transfer through the double helix. The 1.2 A resolution crystal structure of a sequence-specific rhodium intercalator bound to a DNA helix provides a rationale for the sequence specificity of rhodium intercalators. It also explains how intercalation in the center of an oligonucleotide modifies DNA conformation. The rhodium complex intercalates via the major groove where specific contacts are formed with the edges of the bases at the target site. The phi ligand is deeply inserted into the DNA base pair stack. The primary conformational change of the DNA is a doubling of the rise per residue, with no change in sugar pucker from B-form DNA. Based upon the five crystallographically independent views of an intercalated DNA helix observed in this structure, the intercalator may be considered as an additional base pair with specific functional groups positioned in the major groove.     

Securing genome stability by orchestrating DNA repair: removal of radiation-induced clustered lesions in DNA

In addition to double- and single-strand DNA breaks and isolated base modifications, ionizing radiation induces clustered DNA damage, which contains two or more lesions closely spaced within about two helical turns on opposite DNA strands. Post-irradiation repair of single-base lesions is routinely performed by base excision repair and a DNA strand break is involved as an intermediate. Simultaneous processing of lesions on opposite DNA strands may generate double-strand DNA breaks and enhance nonhomologous end joining, which frequently results in the formation of deletions. Recent studies support the possibility that the mechanism of base excision repair contributes to genome stability by diminishing the formation of double-strand DNA breaks during processing of clustered lesions.     

Formation of rings from segments of HeLa-cell nuclear deoxyribonucleic acid

Duplex segments of HeLa-cell nuclear DNA were generated by cleavage with DNA restriction endonuclease from Haemophilus influenzae. About 20-25% of the DNA segments produced, when partly degraded with exonuclease III and annealed, were found to form rings visible in the electron microscope. A further 5% of the DNA segments formed structures that were branched in configuration. Similar structures were generated from HeLa-cell DNA, without prior treatment with restriction endonuclease, when the complementary polynucleotide chains were exposed by exonuclease III action at single-chain nicks. After exposure of an average single-chain length of 1400 nucleotides per terminus at nicks in HeLa-cell DNA by exonuclease III, followed by annealing, the physical length of ring closures was estimated and found to be 0.02-0.1mum, or 50-300 base pairs. An almost identical distribution of lengths was recorded for the regions of complementary base sequence responsible for branch formation. It is proposed that most of the rings and branches are formed from classes of reiterated base sequence with an average length of 180 base pairs arranged intermittenly in HeLa-cell DNA. From the rate of formation of branched structures when HeLa-cell DNA segments were heat-denatured and annealed, it is estimated that the reiterated sequences are in families containing approximately 2400-24000 copies.     

HU protein of Escherichia coli has a role in the repair of closely opposed lesions in DNA

Closely opposed lesions form a unique class of DNA damage that is generated by ionizing radiation. Improper repair of closely opposed lesions could lead to the formation of double strand breaks that can result in increased lethality and mutagenesis. In vitro processing of closely opposed lesions was studied using double-stranded DNA containing a nick in close proximity opposite to a dihydrouracil. In this study we showed that HU protein, an Escherichia coli DNA-binding protein, has a role in the repair of closely opposed lesions. The repair of dihydrouracil is initiated by E. coli endonuclease III and processed via the base excision repair pathway. HU protein was shown to inhibit the rate of removal of dihydrouracil by endonuclease III only when the DNA substrate contained a nick in close proximity opposite to the dihydrouracil. In contrast, HU protein did not inhibit the subsequent steps of the base excision repair pathway, namely the DNA synthesis and ligation reactions catalyzed by E. coli DNA polymerase and E. coli DNA ligase, respectively. The nick-dependent selective inhibition of endonuclease III activity by HU protein suggests that HU could play a role in reducing the formation of double strand breaks in E. coli.     

In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites.

When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS). In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed. The single strand break, a nucleotide gap containing 3' and 5' phosphate groups, was positioned one, three or six nucleotides 5' or 3' to the damage in the complementary DNA strand. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5' or 3' on the opposing strand. When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg. Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5' or 3' to the rAP site.To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3' phosphate termini, insert the "missing" nucleotide, and ligate the nicks, respectively. In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand. However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG. Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced. Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away. Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break.     

Precise binding of single-stranded DNA termini by human RAD52 protein

The human RAD52 protein, which exhibits a heptameric ring structure, has been shown to bind resected double strand breaks (DSBs), consistent with an early role in meiotic recombination and DSB repair. In this work, we show that RAD52 binds single-stranded and tailed duplex DNA molecules via precise interactions with the terminal base. When probed with hydroxyl radicals, ssDNA-RAD52 complexes exhibit a four-nucleotide repeat hypersensitivity pattern. This unique pattern is due to the interaction of RAD52 with either a 5' or a 3' terminus of the ssDNA, is sequence independent and is phased precisely from the terminal nucleotide. Hypersensitivity is observed over approximately 36 nucleotides, consistent with the length of DNA that is protected by RAD52 in nuclease protection assays. We propose that RAD52 binds DNA breaks via specific interactions with the terminal base, leading to the formation of a precisely organized ssDNA-RAD52 complex in which the DNA lies on an exposed surface of the protein. This protein-DNA arrangement may facilitate the DNA-DNA interactions necessary for RAD52-mediated annealing of complementary DNA strands.     

DNA polymerase theta (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct

DNA polymerase theta (pol theta) is a nuclear A-family DNA polymerase encoded by the POLQ gene in vertebrate cells. The biochemical properties of pol theta and of Polq-defective mice have suggested that pol theta participates in DNA damage tolerance. For example, pol theta was previously found to be proficient not only in incorporation of a nucleotide opposite a thymine glycol or an abasic site, but also extends a polynucleotide chain efficiently from the base opposite the lesion. We carried out experiments to determine whether this ability to extend from non-standard termini is a more general property of the enzyme. Pol theta extended relatively efficiently from matched termini as well as termini with A:G, A:T and A:C mismatches, with less descrimination than a well-studied A-family DNA polymerase, exonuclease-free pol I from E. coli. Although pol theta was unable to, by itself, bypass a cyclobutane pyrimidine dimer or a (6-4) photoproduct, it could perform some extension from primers with bases placed across from these lesions. When pol theta was combined with DNA polymerase iota, an enzyme that can insert a base opposite a UV-induced (6-4) photoproduct, complete bypass of a (6-4) photoproduct was possible. These data show that in addition to its ability to insert nucleotides opposite some DNA lesions, pol theta is proficient at extension of unpaired termini. These results show the potential of pol theta to act as an extender after incorporation of nucleotides by other DNA polymerases, and aid in understanding the role of pol theta in somatic mutagenesis and genome instability.     

The post-incision steps of the DNA base excision repair pathway in Escherichia coli: studies with a closed circular DNA substrate containing a single U:G base pair.

The DNA base excision repair pathway is responsible for removal of oxidative and endogenous DNA base damage in both prokaryotes and eukaryotes. This pathway involves formation of an apurinic/apyrimidinic (AP) site in the DNA, which is further processed to restore the integrity of the DNA. In Escherichia coli it has been suggested that the major mode of repair involves replacement of a single nucleotide at the AP site, based on repair synthesis studies using oligonucleotide substrates containing a unique uracil base. The mechanism of the post-incision steps of the bacterial base excision repair pathway was examined using a DNA plasmid substrate containing a single U:G base pair. Repair synthesis carried out by repair-proficient ung, recJ and xon E.coli cell extracts was analyzed by restriction endonuclease cleavage of the DNA containing the uracil lesion. It was found that replacement of the uracil base was always accompanied by replacement of several nucleotides ( approximately 15) 3' of the uracil and this process was absolutely dependent on initial removal of the uracil base by the action of uracil-DNA glycosylase. In contrast to findings with oligonucleotide substrates, replacement of just a single nucleotide at the lesion site was not detected. These results suggest that repair patch length may be substrate dependent and a re-evaluation of the post-incision steps of base excision repair is suggested.     

Roles of base excision repair subpathways in correcting oxidized abasic sites in DNA

Base excision DNA repair (BER) is fundamentally important in handling diverse lesions produced as a result of the intrinsic instability of DNA or by various endogenous and exogenous reactive species. Defects in the BER process have been associated with cancer susceptibility and neurodegenerative disorders. BER funnels diverse base lesions into a common intermediate, apurinic/apyrimidinic (AP) sites. The repair of AP sites is initiated by the major human AP endonuclease, Ape1, or by AP lyase activities associated with some DNA glycosylases. Subsequent steps follow either of two distinct BER subpathways distinguished by repair DNA synthesis of either a single nucleotide (short-patch BER) or multiple nucleotides (long-patch BER). As the major repair mode for regular AP sites, the short-patch BER pathway removes the incised AP lesion, a 5'-deoxyribose-5-phosphate moiety, and replaces a single nucleotide using DNA polymerase (Polbeta). However, short-patch BER may have difficulty handling some types of lesions, as shown for the C1'-oxidized abasic residue, 2-deoxyribonolactone (dL). Recent work indicates that dL is processed efficiently by Ape1, but that short-patch BER is derailed by the formation of stable covalent crosslinks between Ape1-incised dL and Polbeta. The long-patch BER subpathway effectively removes dL and thereby prevents the formation of DNA-protein crosslinks. In coping with dL, the cellular choice of BER subpathway may either completely repair the lesion, or complicate the repair process by forming a protein-DNA crosslink.     

Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase zeta

DNA polymerase zeta (pol zeta), which is required for DNA damage-induced mutagenesis, functions in the error-prone replication of a wide range of DNA lesions. During this process, pol zeta extends from nucleotides incorporated opposite template lesions by other polymerases. Unlike classical polymerases, pol zeta efficiently extends from primer-terminal base pairs containing mismatches or lesions, and it synthesizes DNA with moderate fidelity. Here we describe genetic and biochemical studies of three yeast pol zeta mutant proteins containing substitutions of highly conserved amino acid residues that contact the triphosphate moiety of the incoming nucleotide. The R1057A and K1086A proteins do not complement the rev3Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein. In contrast, the K1061A protein partially complements the rev3Delta mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs. These findings have important implications both for the evolutionary divergence of pol zeta from classical polymerases and for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions.     

Mechanism of Formation of Novel Covalent Drug·DNA Interstrand Cross-Links and Monoadducts by Enediyne Antitumor Antibiotics

The potent enediyne antitumor antibiotic C1027 has been previously reported to induce novel DNA interstrand cross-links and drug monoadducts under anaerobic conditions [Xu et al. (1997) J. Am. Chem. Soc. 119, 1133-1134]. In the present study, we explored the mechanism of formation of these anaerobic DNA lesions. We found that, similar to the aerobic reaction, the diradical species of the activated drug initiates anaerobic DNA damage by abstracting hydrogen atoms from the C4', C1', and C5' positions of the A1, A2, and A3 nucleotides, respectively, in the most preferred 5'GTTA1T/5'ATA2A3C binding sequence. It is proposed that the newly generated deoxyribosyl radicals, which cannot undergo oxidation, likely add back onto the nearby unsaturated ring system of the postactivated enediyne core, inducing the formation of interstrand cross-links, connecting either A1 to A2 or A1 to A3, or drug monoadducts mainly on A2 or A3. Comparative studies with other enediynes, such as neocarzinostatin and calicheamicin gamma1I under similar reaction conditions indicate that the anaerobic reaction process is a kinetically competitive one, depending on the proximity of the drug unsaturated ring system or dioxygen to the sugar radicals and their quenching by other hydrogen sources such as solvent or thiols. It was found that C1027 mainly generates interstrand cross-links, whereas most of the anaerobic lesions produced by neocarzinostatin are drug monoadducts. Calicheamicin gamma1I was found to be less efficient in producing both lesions. The anaerobic DNA lesions induced by enediyne antitumor antibiotics may have important implications for their potent cytotoxicity in the central regions of large tumors, where relative anaerobic conditions prevail.     

Sorting the consequences of ionizing radiation: processing of 8-oxoguanine/abasic site lesions

Clustered DNA damage is a hallmark of ionizing radiation. These complex lesions, composed of any combination of oxidized bases, abasic sites, or strand breaks within one helical turn, create a tremendous challenge for the base excision repair system, which must process the damage without generating cytotoxic double strand breaks (DSB). Clustered lesions affect the DNA incision activity of DNA glycosylases and AP endonucleases. Different levels of enzyme inhibition are dependent on lesion identity, orientation and separation. Very little is known about the simultaneous action of both classes of enzymes, which may lead to the creation of DSB. We have developed a novel substrate system of double-labeled hairpin duplexes, which allows the simultaneous determination of enzyme incision and formation of DBS. We use this system to study the processing of four clustered 8-oxoguanine/abasic site lesions by purified mouse Ogg1, human Ape1 and mouse embryonic stem cell nuclear extracts. Ape1 activity is least affected by the presence of a nearby oxidized base. In contrast, an abasic site inhibits the glycosylase and lyase activities of Ogg1 in an orientation-dependent manner. The combined action of both enzymes leads to the preferential formation of DSB with 5'-overhang ends. Processing of clusters by nuclear extracts displayed similar patter of enzyme inhibition and the same preference for avoiding double strand breaks with 3'-overhang ends.     

Conformational changes of the phenyl and naphthyl isocyanate-DNA adducts during DNA replication and by minor groove binding molecules

DNA lesions produced by aromatic isocyanates have an extra bulky group on the nucleotide bases, with the capability of forming stacking interaction within a DNA helix. In this work, we investigated the conformation of the 2′-deoxyadenosine and 2′-deoxycytidine derivatives tethering a phenyl or naphthyl group, introduced in a DNA duplex. The chemical modification experiments using KMnO₄ and 1-cyclohexyl-3 -(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate have shown that the 2′-deoxycytidine lesions form the base pair with guanine while the 2′-deoxyadenosine lesions have less ability of forming the base pair with thymine in solution. Nevertheless, the kinetic analysis shows that these DNA lesions are compatible with DNA ligase and DNA polymerase reactions, as much as natural DNA bases. We suggest that the adduct lesions have a capability of adopting dual conformations, depending on the difference in their interaction energies between stacking of the attached aromatic group and base pairing through hydrogen bonds. It is also presented that the attached aromatic groups change their orientation by interacting with the minor groove binding netropsin, distamycin and synthetic polyamide. The nucleotide derivatives would be useful for enhancing the phenotypic diversity of DNA molecules and for exploring new non-natural nucleotides.     

OFF-to-ON type fluorescent probe for the detection of 8-oxo-dG in DNA by the Adap-masked ODN probe

We have recently reported that Adap (adenosine-1,3-diazaphenoxazine) is an artificial nucleoside analogue for the specific recognition by multiple hydrogen bonding and that its fluorescence is selectively quenched with 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA. We now report the development of a new OFF-to-ON type FRET probe, in which one strand contains Adap and another contains natural nucleotides for the formation of a less stable double strand. Each strand was labeled with Cy3 or BHQ2 at the 5'-end or 3'-end, respectively. It was expected in this system that fluorescence of the duplex probe is first quenched by FRET, but the target DNA strand containing 8-oxo-dG at the complementary site of Adap would enhance the displacement reaction of the less stable duplex probe that results in the fluorescence recovery. The results showed that the duplex probe containing the Adap-T base pair exhibited a complete discrimination between 8-oxo-dG and dG in DNA by fluorescence enhancement.     

On loop folding in nucleic acid hairpin-type structures

In a series of studies, combining NMR, optical melting and T-jump experiments, it was found that DNA hairpins display a maximum stability when the loop part of the molecule comprises four or five nucleotide residues. This is in contrast with the current notion based on RNA hairpin studies, from which it had been established that a maximum hairpin stability is obtained for six or seven residues in the loop. Here we present a structural model to rationalize these observations. This model is based on the notion that to a major extent base stacking interactions determine the stability of nucleic acid conformations. The model predicts that loop folding in RNA is characterized by an extension of the base stacking at the 5'-side of the double helix by five or six bases; the remaining gap can then easily be closed by two nucleotides. Conversely, loop folding in DNA is characterized by extending base stacking at the 3'-side of the double helical stem by two or three residues; again bridging of the remaining gap can then be achieved by one or two nucleotides. As an example of loop folding in RNA the anticodon loop of yeast tRNAPhe is discussed. For the DNA hairpin formed by d(ATCCTAT4TAGGAT) it is shown that the loop structure obtained from molecular mechanics calculations obeys the above worded loop folding principles.     

Stability of 3' double nucleotide overhangs that model the 3' ends of siRNA

Thermodynamic parameters are reported for duplex formation in 1 M NaCl for 16 RNA sequences, each containing a core tetramer duplex, GGCC, and a 3' overhang consisting of two bases. The results indicate additional double-helical stability is conferred by the double 3' terminal overhang relative to the single 3' terminal overhang. A nearest-neighbor analysis of the data indicates that the free energy contribution at 37 degrees C of the second base in the double 3' terminal overhang varies from 0 to 0.7 kcal/mol. The second base in the 3' double overhang can contribute nearly the same stability to a duplex as a base pair or a 3' dangling overhang. Stability contribution of a dangling base, two nucleotides removed from the 3' end of a duplex, is dependent upon both the identity of the base as well as that of the dangling base that it neighbors. A second dangling base only increases the stability of the duplex when it is neighboring a 3' purine dangling nucleotide. Furthermore, a second dangling pyrimidine provides a greater contribution to duplex stability than a purine. A nearest-neighbor model was developed to predict the influence of 3' double overhang on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against six sequences with different core duplexes.