Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIβ. We show that topo IIβ is a DNA-dependent ATPase that appears to fit Michaelis–Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k_cat for ATP hydrolysis by human DNA topo IIβ in the presence of DNA is 2.25 s^–1. We have characterised a topo IIβ derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the k_cat for ATP hydrolysis by 7-fold, to 0.32 s^–1, while not significantly altering the apparent K_m. The specificity constant for the interaction between ATP and topo IIβ (k_cat/K_mapp) showed a 90% reduction for βS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIβ. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.     

Esp1396I restriction-modification system: structural organization and mode of regulation

Esp1396I restriction–modification (RM) system recognizes an interrupted palindromic DNA sequ ence 5′-CCA(N)₅TGG-3′. The Esp1396I RM system was found to reside on pEsp1396, a 5.6 kb plasmid naturally occurring in Enterobacter sp. strain RFL1396. The nucleotide sequence of the entire 5622 bp pEsp1396 plasmid was determined on both strands. Identified genes for DNA methyltransferase (esp1396IM) and restriction endonuclease (esp1396IR) are transcribed convergently. The restriction endonuclease gene is preceded by the small ORF (esp1396IC) that possesses a strong helix-turn-helix motif and resembles regulatory proteins found in PvuII, BamHI and few other RM systems. Gene regulation studies revealed that C.Esp1396I acts as both a repressor of methylase expression and an activator of regulatory protein and restriction endonuclease expression. Our data indicate that C protein from Esp1396I RM system activates the expression of the Enase gene, which is co-transcribed from the promoter of regulatory gene, by the mechanism of coupled translation.     

An essential DnaB helicase of Bacillus anthracis: identification, characterization, and mechanism of action

We have described a novel essential replicative DNA helicase from Bacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB helicase (DnaB_BA) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaB_BA displayed distinct enzymatic and kinetic properties. DnaB_BA has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5′→3′ DNA helicase activity. The stimulation of ATPase activity appeared to be a function of the length of the ssDNA template rather than of ssDNA binding alone. The highest specific activity was observed with M13mp19 ssDNA. The results presented here indicated that the ATPase activity of DnaB_BA was coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaB_BA demonstrated a strong DNA helicase activity that required ATP or dATP. Therefore, DnaB_BA has an attenuated ATPase activity and a highly active DNA helicase activity. Based on the ratio of DNA helicase and ATPase activities, DnaB_BA is highly efficient in DNA unwinding and its coupling to ATP consumption.     

Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 5′-CTGATG(N)_25-26/27-28-3′. Endonuclease activity requires a substrate with two copies of the recognition site in head-to-head repeat and is dependent on a low level of ATP hydrolysis (~40 ATP/site/min). Cleavage occurs at just one of the two sites and results in a staggered cut 25–26 nt downstream of the top strand sequence to generate a two base 5′-protruding end. Methylation of the site occurs on one strand only at the first adenine of 5′-CATCAG-3′. Therefore, PstII has characteristic Type III restriction enzyme activity as exemplified by EcoPI or EcoP15I. Moreover, sequence asymmetry of the PstII recognition site in the T7 genome acts as an historical imprint of Type III restriction activity in vivo. In contrast to other Type I and III enzymes, PstII has a more relaxed nucleotide specificity and can cut DNA with GTP and CTP (but not UTP). We also demonstrate that PstII and EcoP15I cannot interact and cleave a DNA substrate suggesting that Type III enzymes must make specific protein–protein contacts to activate endonuclease activity.     

Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45°C, and an optimal divalent-cation concentration of 5 mM MgCl₂. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The K_m values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 μM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3′-to-5′ direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5′ or 3′ tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3′ tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.     

Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5′-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5′-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA–R.PabI reaction intermediates can be trapped by NaBH₄ reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.     

The isolation of strand-specific nicking endonucleases from a randomized SapI expression library

The Type IIS restriction endonuclease SapI recognizes the DNA sequence 5′-GCTCTTC-3′ (top strand by convention) and cleaves downstream (N1/N4) indicating top- and bottom-strand spacing, respectively. The asymmetric nature of DNA recognition presented the possibility that one, if not two, nicking variants might be created from SapI. To explore this possibility, two parallel selection procedures were designed to isolate either top-strand nicking or bottom-strand nicking variants from a randomly mutated SapI expression library. These procedures take advantage of a SapI substrate site designed into the expression plasmid, which allows for in vitro selection of plasmid clones possessing a site-specific and strand-specific nick. A procedure designed to isolate bottom-strand nicking enzymes yielded Nb.SapI-1 containing a critical R420I substitution near the end of the protein. The top-strand procedure yielded several SapI variants with a distinct preference for top-strand cleavage. Mutations present within the selected clones were segregated to confirm a top-strand nicking phenotype for single variants Q240R, E250K, G271R or K273R. The nature of the amino acid substitutions found in the selected variants provides evidence that SapI may possess two active sites per monomer. This work presents a framework for establishing the mechanism of SapI DNA cleavage.     

ATPase Mechanism of the 5′-3′ DNA Helicase,RecD2: EVIDENCE FOR A PRE-HYDROLYSIS CONFORMATION CHANGE*

The superfamily 1 helicase, RecD2, is a monomeric, bacterial enzyme with a role in DNA repair, but with 5′-3′ activity unlike most enzymes from this superfamily. Rate constants were determined for steps within the ATPase cycle of RecD2 in the presence of ssDNA. The fluorescent ATP analog, mantATP (2′(3′)-O-(N-methylanthraniloyl)ATP), was used throughout to provide a complete set of rate constants and determine the mechanism of the cycle for a single nucleotide species. Fluorescence stopped-flow measurements were used to determine rate constants for adenosine nucleotide binding and release, quenched-flow measurements were used for the hydrolytic cleavage step, and the fluorescent phosphate biosensor was used for phosphate release kinetics. Some rate constants could also be measured using the natural substrate, ATP, and these suggested a similar mechanism to that obtained with mantATP. The data show that a rearrangement linked to Mg²⁺ coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA. This is also shown here for the PcrA 3′-5′ helicase and so may be a general mechanism governing superfamily 1 helicases. The mechanism accounts for the tight coupling between translocation and ATPase activity.     

ATPase Activity of the Type IC Restriction-Modification SystemEcoR124II

We have investigated the ATPase activity of the type IC restriction-modification (R – M) systemEcoR124II. As with all type I R – M systemsEcoR 124II requires ATP hydrolysis to cut DNA. We determined theK_Mfor ATP to be 10⁻⁵to 10⁻⁴M. By measuring ATP hydrolysis under different conditions and by simultaneously monitoring DNA restriction, methylation and ATP hydrolysis we propose that the order of events during restriction is: (1) binding ofEcoR124II to a non-methylated recognition sequence, (2) start of DNA-dependent ATP hydrolysis which continues even after restriction is complete, (3) restriction of DNA, (4) methylation of the product. Non-cleavable DNA substrates, such as recognition site containing oligonucleotides, also support ATP hydrolysis. Methylation can also occur prior to ATP hydrolysis and prevent DNA degradation.     

Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily

The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5′-GGTAC^C-3′ in the presence of Mg²⁺ as shown generating 3′ four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a ββα-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD…D/EXK superfamily of nucleases, instead is a member of the HNH superfamily.     

Molecular mechanism of sequence-dependent stability of RecA filament

RecA is a DNA-dependent ATPase and mediates homologous recombination by first forming a filament on a single-stranded (ss) DNA. RecA binds preferentially to TGG repeat sequence, which resembles the recombination hot spot Chi (5′-GCTGGTGG-3′) and is the most frequent pattern (GTG) of the codon usage in Escherichia coli. Because of the highly dynamic nature of RecA filament formation, which consists of filament nucleation, growth and shrinkage, we need experimental approaches that can resolve each of these processes separately to gain detailed insights into the molecular mechanism of sequence preference. By using a single-molecule fluorescence assay, we examined the effect of sequence on individual stages of nucleation, monomer binding and dissociation. We found that RecA does not recognize the Chi sequence as a nucleation site. In contrast, we observed that it is the reduced monomer dissociation that mainly determines the high filament stability on TGG repeats. This sequence dependence of monomer dissociation is well-correlated with that of ATP hydrolysis, suggesting that DNA sequence dictates filament stability through modulation of ATP hydrolysis.     

Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP

The 2′,3′-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H⁺-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH₄. The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K_i of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s⁻¹. The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [³H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.     

The Ser176 of T4 endonuclease IV is crucial for the restricted and polarized dC-specific cleavage of single-stranded DNA implicated in restriction of dC-containing DNA in host Escherichia coli

Endonuclease (Endo) IV encoded by denB of bacteriophage T4 is an enzyme that cleaves single-stranded (ss) DNA in a dC-specific manner. Also the growth of dC-substituted T4 phage and host Escherichia coli cells is inhibited by denB expression presumably because of the inhibitory effect on replication of dC-containing DNA. Recently, we have demonstrated that an efficient cleavage by Endo IV occurs exclusively at the 5′-proximal dC (dC₁) within a hexameric or an extended sequence consisting of dC residues at the 5′-proximal and the 3′-proximal positions (dCs tract), in which a third dC residue within the tract affects the polarized cleavage and cleavage rate. Here we isolate and characterize two denB mutants, denB(W88R) and denB(S176N). Both mutant alleles have lost the detrimental effect on the host cell. Endo IV(W88R) shows no enzymatic activity (<0.4% of that of wild-type Endo IV). On the other hand, Endo IV(S176N) retains cleavage activity (17.5% of that of wild-type Endo IV), but has lost the polarized and restricted cleavage of a dCs tract, indicating that the Ser¹⁷⁶ residue of Endo IV is implicated in the polarized cleavage of a dCs tract which brings about a detrimental effect on the replication of dC-containing DNA.     

A novel single-stranded DNA-specific 3′–5′ exonuclease,Thermus thermophilus exonuclease I,is involved in several DNA repair pathways

Single-stranded DNA (ssDNA)-specific exonucleases (ssExos) are expected to be involved in a variety of DNA repair pathways corresponding to their cleavage polarities; however, the relationship between the cleavage polarity and the respective DNA repair pathways is only partially understood. To understand the cellular function of ssExos in DNA repair better, genes encoding ssExos were disrupted in Thermus thermophilus HB8 that seems to have only a single set of 5′–3′ and 3′–5′ ssExos unlike other model organisms. Disruption of the tthb178 gene, which was expected to encode a 3′–5′ ssExo, resulted in significant increase in the sensitivity to H₂O₂ and frequency of the spontaneous mutation rate, but scarcely affected the sensitivity to ultraviolet (UV) irradiation. In contrast, disruption of the recJ gene, which encodes a 5′–3′ ssExo, showed little effect on the sensitivity to H₂O₂, but caused increased sensitivity to UV irradiation. In vitro characterization revealed that TTHB178 possessed 3′–5′ ssExo activity that degraded ssDNAs containing deaminated and methylated bases, but not those containing oxidized bases or abasic sites. Consequently, we concluded that TTHB178 is a novel 3′–5′ ssExo that functions in various DNA repair systems in cooperation with or independently of RecJ. We named TTHB178 as T. thermophilus exonuclease I.     

A novel nuclease-ATPase (Nar71) from archaea is part of a proposed thermophilic DNA repair system

We have identified a novel structure-specific nuclease in highly fractionated extracts of the thermophilic archaeon Methanothermobacter thermautotrophicus (Mth). The 71 kDa protein product of open reading frame mth1090 is a nuclease with ATPase activity, which we call Nar71 (Nuclease-ATPase in Repair, 71 kDa). The nar71 gene is located in a gene neighbourhood proposed by genomics to encode a novel DNA repair system conserved in thermophiles. The biochemical characterization of Nar71 presented here is the first analysis from within this neighbourhood, and it supports the insight from genomics. Nuclease activity of Nar71 is specific for 3′ flaps and flayed duplexes, targeting single-stranded DNA (ssDNA) regions. This activity requires Mg²⁺ or Mn²⁺ and is greatly reduced in ATP. In ATP, Nar71 displaces ssDNA, also with high specificity for 3′ flap and flayed duplex DNA. Strand displacement is weak compared with nuclease activity, but in ATPγS it is abolished, suggesting that Nar71 couples ATP hydrolysis to DNA strand separation. ATPase assays confirmed that Nar71 is stimulated by ssDNA, though not double-stranded DNA. Mutation of Lys-117 in Nar71 abolished ATPase and nuclease activity, and we describe a separation-of-function mutant (K68A) that has lost ATPase activity but retains nuclease activity. A model of possible Nar71 function in DNA repair is presented.     

Werner syndrome exonuclease catalyzes structure-dependent degradation of DNA

Werner syndrome (WS) is an autosomal recessive disease characterized by early onset of many features of aging, by an unusual spectrum of cancers, and by genomic instability. The WS protein (WRN) possesses 3′→5′ DNA helicase and associated ATPase activities, as well as 3′→5′ DNA exonuclease activity. Currently, WRN is the only member of the widely distributed RecQ DNA helicase family with documented exonuclease activity. It is not known whether deficiency of the exonuclease or helicase/ATPase activities of WRN, or all of them, is responsible for various elements of the WS phenotype. WRN exonuclease has limited homology to Escherichia coli RNaseD, a tRNA processing enzyme. We show here that WRN preferentially degrades synthetic DNA substrates containing alternate secondary structures, with an exonucleolytic mode of action suggestive of RNaseD. We present evidence that structure-dependent binding of WRN to DNA requires ATP binding, while DNA degradation requires ATP hydrolysis. Apparently, the exonuclease and ATPase act in concert to catalyze structure-dependent DNA degradation. We propose that WRN protein functions as a DNA processing enzyme in resolving aberrant DNA structures via both exonuclease and helicase activities.     

Electron attachment-induced DNA single-strand breaks at the pyrimidine sites

To elucidate the contribution of pyrimidine in DNA strand breaks caused by low-energy electrons (LEEs), theoretical investigations of the LEE attachment-induced C_3′–O_3′, and C_5′–O_5′ σ bond as well as N-glycosidic bond breaking of 2′-deoxycytidine-3′,5′-diphosphate and 2′-deoxythymidine-3′,5′-diphosphate were performed using the B3LYP/DZP++ approach. The base-centered radical anions are electronically stable enough to assure that either the C–O or glycosidic bond breaking processes might compete with the electron detachment and yield corresponding radical fragments and anions. In the gas phase, the computed glycosidic bond breaking activation energy (24.1 kcal/mol) excludes the base release pathway. The low-energy barrier for the C_3′–O_3′ σ bond cleavage process (∼6.0 kcal/mol for both cytidine and thymidine) suggests that this reaction pathway is the most favorable one as compared to other possible pathways. On the other hand, the relatively low activation energy barrier (∼14 kcal/mol) for the C_5′–O_5′ σ bond cleavage process indicates that this bond breaking pathway could be possible, especially when the incident electrons have relatively high energy (a few electronvolts). The presence of the polarizable medium greatly increases the activation energies of either C–O σ bond cleavage processes or the N-glycosidic bond breaking process. The only possible pathway that dominates the LEE-induced DNA single strands in the presence of the polarizable surroundings (such as in an aqueous solution) is the C_3′–O_3′ σ bond cleavage (the relatively low activation energy barrier, ∼13.4 kcal/mol, has been predicted through a polarizable continuum model investigation). The qualitative agreement between the ratio for the bond breaks of C_5′–O_5′, C_3′–O_3′ and N-glycosidic bonds observed in the experiment of oligonucleotide tetramer CGAT and the theoretical sequence of the bond breaking reaction pathways have been found. This consistency between the theoretical predictions and the experimental observations provides strong supportive evidences for the base-centered radical anion mechanism of the LEE-induced single-strand bond breaking around the pyrimidine sites of the DNA single strands.     

Unidirectional translocation from recognition site and a necessary interaction with DNA end for cleavage by Type III restriction enzyme

Type III restriction enzymes have been demonstrated to require two unmethylated asymmetric recognition sites oriented head-to-head to elicit double-strand break 25–27 bp downstream of one of the two sites. The proposed DNA cleavage mechanism involves ATP-dependent DNA translocation. The sequence context of the recognition site was suggested to influence the site of DNA cleavage by the enzyme. In this investigation, we demonstrate that the cleavage site of the R.EcoP15I restriction enzyme does not depend on the sequence context of the recognition site. Strikingly, this study demonstrates that the enzyme can cleave linear DNA having either recognition sites in the same orientation or a single recognition site. Cleavage occurs predominantly at a site proximal to the DNA end in the case of multiple site substrates. Such cleavage can be abolished by the binding of Lac repressor downstream (3′ side) but not upstream (5′ side) of the recognition site. Binding of HU protein has also been observed to interfere with R.EcoP15I cleavage activity. In accordance with a mechanism requiring two enzyme molecules cooperating to elicit double-strand break on DNA, our results convincingly demonstrate that the enzyme translocates on DNA in a 5′ to 3′ direction from its recognition site and indicate a switch in the direction of enzyme motion at the DNA ends. This study demonstrates a new facet in the mode of action of these restriction enzymes.     

Synthesis,characterization and solution structure of tethered oligonucleotides containing an internal 3'-phosphoglycolate, 5'-phosphate gapped lesion

Bleomycins (BLMs) are antitumor antibiotics that in the presence of iron and oxygen mediate DNA damage by 4′-hydrogen atom abstraction of pyrimidines 3′ to guanines. The resulting 4′-deoxyribose radicals can be trapped by O₂ and ultimately result in the formation of base-propenal and gapped DNA with 3′-phosphoglycolate (3′-PG) and 5′-phosphate (5′-P) ends. The role of this lesion in triggering double-strand cleavage of duplex DNA by a single BLM molecule and the mechanism by which this lesion is repaired in vivo remain unsolved problems. The structure of these lesions is an essential step in addressing both of these problems. Duplex DNAs (13mers containing tethered hexaethylene glycol linkers) with GTAC and GGCC cleavage sites have been synthesized in which gaps containing 3′-PG and 5′-P ends at the sites of BLM cleavage have been inserted. The former sequence represents a hot spot for double-strand cleavage, while the latter is a hot spot for single-strand cleavage. Analytical methods to characterize the lesioned products have been developed. These oligonucleotides have been examined using 2D NMR methods and molecular modeling. The studies reveal that the lesioned DNAs are B-form and the 3′-PG and 5′-P are extrahelical. The base opposite the gap and the base pairs adjacent to the gap remain well stacked in the DNA duplex. Titrations of the lesioned GGCC oligomer with HOO-CoBLM leads to a mixture of complexes, in contrast to results of a similar titration with the lesioned GTAC oligomer.