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Nucleic Acids Research, 2002, Vol. 30, No. 24 5497-5508
© 2002 Oxford University Press

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

Hans-Dieter Junker1, Silvia T. Hoehn1, Richard C. Bunt1, Vasilios Marathius1, Jingyang Chen1, Christopher J. Turner3 and JoAnne Stubbe*,1,2

1 Department of Chemistry, 2 Department of Biology and 3 Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

*To whom correspondence should be addressed. Tel: +1 617 253 1814; Fax: +1 617 258 7247; Email: stubbe{at}mit.edu

Received August 13, 2002; Revised and Accepted October 24, 2002PDB nos 1G5K, 1GJ1, 1N0O, 1N0K


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The cytotoxic effects of a variety of antitumor antibiotics such as the bleomycins (BLMs) and enediynes are thought to be associated with their ability to bind to DNA and produce sequence-specific single-strand (ss) and double-strand (ds) breaks in the DNA by damaging the deoxyribose backbone. Even though these natural products differ in the details of their mechanism of action, they share the ability to produce a 4'-deoxyribose radical that can lead to two types of damage (Fig. 1) (1). If the radical is trapped with O2 the DNA strand is cleaved directly, generating a gapped duplex containing 3'-phosphoglycolate (3'-PG) and 5'-phosphate (5'-P) ends and a base-propenal. Alternatively, under low O2 tensions, a 4'-ketoabasic site with an intact DNA strand and a free nucleic acid base results (2–4). Our laboratory has focused recently on understanding the mechanism of BLM-mediated ds breaks and the role of the 3'-PG lesion in this process (5–7). Double-strand breaks are believed to be the major cause of cytotoxicity of the BLMs due to the difficulty of their repair (8–10). Our interest in the mechanism of action of these antitumor agents has led us to study the mechanisms of repair of the lesions generated by these natural products. This paper presents the first insight into the structures of duplex oligomeric DNAs containing a GXAC and GGXC sequence (where X is a gapped 3'-PG/5'-P lesion) using 2D NMR methods and molecular modeling. This study is the starting point for understanding the basis of recognition of damaged sugars and the mechanism of their repair.



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  		Figure 1. Postulated mechanism of BLM-mediated ss and ds cleavage pathways. Pathway A requires O2 over and above the O2 required to generate activated BLM and leads to the generation of a gapped 3'-PG and 5'-P lesion and base-propenal. An intermediate in this pathway is essential for reactivation of BLM so that a second cleavage event leading to ds cleavage can occur without BLM dissociating from the DNA. Pathway B results in nucleic acid base release and a 4'-ketoabasic site lesion in intact DNA.

 
Base excision repair (BER) is an important pathway in the repair of ss damage to the deoxyribose of DNA caused by BLM in the presence of its required cofactors Fe2+ and O2 (11–13). In humans the apyrimidinic/apurinic endonuclease APE1 (also known as HAP1, Ref-1 or Apex) is the protein in the BER pathway that is responsible for the recognition and repair of abasic sites (14,15). This enzyme is also inducible in response to BLM (16,17) and can repair the 4'-ketoabasic sites and 3'-PG lesions generated by BLM (11,12,18). The efficiency of removal of the 4'-ketoabasic site in one sequence context is similar to abasic sites (13).

The 3'-PG lesion repair by APE1 in vitro is slow and depends on the location of the damage within the duplex, and thus the exact role this enzyme plays in its repair in vivo is not known. 3'-PGs are removed from oligonucleotide substrates with an efficiency that is 70- to 400-fold lower than removal of abasic sites (12,19). In mammalian cell extracts at least two additional 3'-phosphodiesterase activities have been detected, but not isolated due to their instability (20,21). Furthermore, recent reports suggest that APE1 is one of a complex of enzymes involved in the BER pathway and in such a complex the kinetics could be very different (22).

The kinetics and mechanism by which activated BLM generates a gapped 3'-PG next to a 5'-P have been investigated in detail (Fig. 1). Activated BLM abstracts the H4' of a pyrimidine 3' to a guanine (recently reviewed in 2,3,23,24). O2 reacts with the resulting C4' radical which is reduced to a 4'-hydroperoxide. This species ultimately leads to strand scission generating a 3'-PG end with a t1/2 = 2.5–5 min at 4°C. Kinetic studies in one system reveal that 3'-PG production precedes base-propenal release and 5'-P formation which occur with a t1/2 of 6.7 ± 0.3 min at 4°C (25). If these in vitro experiments are indicative of the reaction in vivo and the rates are sequence independent, then repair enzymes may encounter an intermediate in the strand scission pathway consisting of a 3'-PG adjacent to a fragment of deoxyribose with base still attached, the precursor to the 5'-P end (Fig. 1). However, most DNA damage does not result in ds lesions (ds:ss cleavage ratios are typically 1:10–1:20) (8,26,27) and the average kinetic parameters measured in vitro may not be representative of the lesions that result in ds breaks. Thus the actual lesion(s) that leads to ds cleavage and the lesions recognized and repaired in vivo have not been identified.

APE1 has been crystallized in the presence of an oligonucleotide containing a tetrahydrofuran (THF) analog of an abasic site (28). The binding pocket for the damaged deoxyribose is small and cannot accommodate a base. Consistent with this model, APE1 does not bind to nicked DNA containing a 3'-OH and a 5'-P mononucleotide (12). APE1 does, on the other hand, recognize, in addition to a modified deoxyribose, a space bracketed by duplex DNA (19). Thus structural data suggests that APE1 could recognize a 3'-PG and a gapped 5'-P.

Double-strand cleavage of DNA by BLM occurs only when a 3'-PG next to a 5'-P is generated initially at the ss cleavage site. Evidence suggests that a single molecule of activated BLM can cause both cleavage events without dissociation from the DNA. Based on our recent NMR studies with the hydroperoxide of cobalt BLM (HOO-CoBLM) bound to a hot spot for ds cleavage, a model involving the importance of the bithiazole tail of BLM in the reorganization of the BLM from one strand to the second has been proposed (7,29). The trigger that causes the BLM to reorganize, however, remains elusive. Since BLM-induced ds cleavage only occurs at 3'-PG ends, it is possible that either the 3'-PG next to the 5'-P, or a precursor thereof, triggers the reorganization of the bithiazole tail, which ultimately leads to ds cleavage.

Our laboratory has long been interested in the mechanism of BLM-mediated ss and ds cleavage of DNA. As a starting point to study ds cleavage catalyzed by BLM and the mechanism of its repair, we have synthesized gapped duplex DNAs with 3'-PG next to a 5'-P (denoted by X) in the sequence contexts d(CCAAAGXACTGGG) (1a) and d(CCAAGGXCTTGGG) (1b). We have previously shown that GTAC is a hot spot for BLM-induced ds cleavage and that GGCC is a hot spot for ss cleavage and not ds cleavage (6). To ensure the stability of the oligomeric duplex, hexaethylene glycol spacers have been used to connect C1 to G26 and G13 to C14 in the double hairpin of 1a and 1b (Fig. 2). In this paper we describe a general synthesis and characterization for PG lesions. The solution structure of 1a and 1b, established by 2D NMR experiments and molecular modeling, provides the first structural models of 3'-PG and 5'-P gapped lesions.



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  		Figure 2. Hairpin oligonucleotides 1a and 1b. Hexaethylene glycol spacers connect the two strands of the duplex. In 1a, GTAC is the hot spot for ds cleavage and in 1b GGCC is the hot spot for ss cleavage.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Solid phase oligonucleotide synthesis
Compounds 5a and 5b (Fig. 3) were synthesized on the 1 µmol scale using deoxyribonucleotide phosphoramidites and reagents from Perkin Elmer and an ABI 392 DNA synthesizer (30,31). The synthesis of 5a is reported in detail and that of 5b followed a similar protocol. The 3-[(4',4-dimethoxytrityloxy)glyceryl-1-succinoyl]-long chain alkylamino controlled pore glass columns (2; Fig. 3), the spacer 9-O-dimethoxytrityl-hexaethylene glycol 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (3; Fig. 3), [3-(4,4'-dimethoxytrityloxy)-2,2-dicarboxyethyl]propyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (4) and the dicyanoimidazole solution (0.25 M) were purchased from Glen Research. The following changes from the standard automatic synthesis protocol of ABI were made: (i) synthesis was carried out using a 0.25 M dicyanoimidazole solution in place of the tetrazole activator; (ii) a coupling time of 325 s instead of 25 s was used in the synthesis cycle for 3 (Fig. 3); (iii) the coupling time using 4 was extended to 480 s and the capping procedure in this cycle was omitted.



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  		Figure 3. Synthesis of 1a and 1b. (Step a) (i) 33% NH3, 2 h, room temperature; (ii) 33% NH3, 8 h, 55°C. (Step b) (i) 80% acetic acid, 5 h, room temperature; (ii) 33% NH3, 1.5 h, room temperature. (Step c) NaIO4, 2.5 h, 0°C. (Step d) NaClO2, 3 h, room temperature.

 
Deprotection and reverse phase HPLC purification
5a was cleaved from the solid support (2 h, 20°C) and deprotected (8 h, 55°C) with 5 ml of concentrated (33%) aqueous ammonia. The results of 10 (1 µmol scale) syntheses were pooled and concentrated in vacuo. The resulting viscous oil was purified by reverse phase HPLC using a semi-preparative Alltech Econosil C18 column (10 x 250 mm) and a linear gradient of 10–50% acetonitrile in 0.1 M triethylammonium acetate (pH 7.3) over 25 min, followed by a linear gradient of 50–100% acetonitrile over 10 min (flow rate 3 ml/min). The retention time of the product was 20– 23 min. Fractions containing this product were pooled, concentrated to a colorless gum, dissolved in 5 ml of 80% acetic acid and incubated at room temperature for 5 h. The acetic acid was then removed in vacuo and the residue was dissolved in 7 ml of concentrated aqueous ammonia and incubated for 90 min at room temperature. The ammonia was evaporated in vacuo to give a white solid, 6a. The residue was taken up in 4 ml of water, filtered and used directly in the next step. The isolated yield of 6a was 2.3 µmol, based on the calculated extinction coefficient ({epsilon}260 = 1.9 x 105/M/cm). The isolated yield of 6b was 1.3 µmol.

Oxidation of 6 (Fig. 3)
Conversion of 6a to 1a was carried out by a modification of the procedure of Urata and Akagi (32). Sodium periodate (0.2 M, 0.5 ml) was added at 0°C to 6a (2.3 µmol, 4 ml of water) and the mixture stirred for 2.5 h. The reaction was then quenched at 0°C by addition of methionine (0.2 M, 0.72 ml) and stirred for an additional 30 min. The pH was then adjusted to 5 by addition of NaH2PO4 (0.31 M, 1.6 ml) and acetic acid (20– 50 µl). To this solution of 7a (2.3 µmol, 6.4 ml of water) was added NaClO2 (0.2 M, 90 µl) at room temperature (33). The oxidation reaction was monitored by ion exchange HPLC using a Dionex DNApac analytical ion exchange column (4 x 250 mm) using a linear gradient of 35–50% B over 30 min (flow rate 1.0 ml/min). Buffer A contained 25 mM sodium acetate in 90% H2O/10% acetonitrile (pH 6.0); buffer B contained buffer A and 1.0 M NaCl. Compounds 6a, 7a and 1a eluted with retention times of ~22.5, 23 and ~25 min, respectively. Compounds 6b, 7b and 1b eluted with retention times of 24.1, 24.7 and 26.9 min. The reaction was quenched after ~95% conversion (~3 h) with Na2SO3 (0.2 M, 90 µl) and the pH was adjusted to ~6.5 with 0.2 M Na2HPO4. The crude product (~75% pure) was dialyzed (Spectrum dialysis tubing, mol. wt cut-off 1000 g/mol) against 2 l of 5 mM sodium phosphate (pH 6.9) buffer for 36 h at 4°C.

Purification of 1a
Compound 1a was purified by HPLC using a semi-preparative Dionex Nucleopac PA 100 column (9.0 x 250 mm). The same gradient as described above was applied (flow rate 4.0 ml/min). The product was dialyzed at 4°C against 2 x 2 l of 5 mM sodium phosphate (pH 6.9) buffer for 48 h. The isolated yield of 1a was 54% (1.2 µmol). The isolated yield of 1b, prepared by an identical procedure, was 0.72 µmol, 54%.

Electrospray mass spectroscopy (ES-MS)
Mass spectra were obtained in the MIT Biopolymer Laboratory using an ABI 140c high performance liquid chromatograph (HPLC) interfaced with a PE Sciex API365 mass spectrometer. The electrospray source was adjusted to a spray voltage of ~3.5 kV and a capillary temperature of 125–200°C. 1a was exchanged into a volatile buffer using an ABI C18 RP300 column (10 x 100 mm) and eluted using a linear gradient of 7–17% acetonitrile in 0.1 M triethylammonium acetate (pH 7.3) over 45 min (flow rate 50 µl/min). The chromatogram showed a single peak with a retention time of 8.5 min and a molecular mass of 8574.1 (± 1.4) g/mol (calculated: 8573.6 g/mol). For 1b a molecular mass of 8590.2 g/mol (calculated: 8590.6 g/mol) was observed.

Characterization of 1a by enzymatic digestion
Compound 1a (0.82 nmol, 0.04 mM) was mixed with snake venom phosphodiesterase (SVPDE) from Crotalus adamanteus [specific activity 20 U/mg, 0.06 U (2 µl); Pharmacia], DNase from bovine pancreas [specific activity 2900 U/mg, 3.0 U (2 µl); Sigma] and buffer [10 mM Tris–HCl (pH 7.5), 4 mM MgCl2, 76 µl] leading to a final volume of 100 µl. The mixture was incubated for 20 h at 37°C. Twenty percent of the digestion mixture was analyzed by ion exchange HPLC using a Dionex Nucleopac analytical column (4 x 250 mm) and a linear gradient of 0–50% B over 20 min (flow rate 1.0 ml/min). Buffer A contained 0.025 M sodium acetate (pH 6.0) in 90% H2O/10% acetonitrile; buffer B contained buffer A and 1.0 M NaCl. Two peaks in the HPLC trace were detected and assigned to 3' modified nucleotides. Peak A (retention time 5.5 min) was desalted and assigned to 5'-phospho-deoxyguanosine-3'-phosphohexakis(ethylene glycol), based on ES-MS (observed: 691.8 g/mol, calculated: 691.5 g/mol). Peak B (retention time 9.2 min) had an identical retention time and UV/vis spectrum to 5'-phospho-deoxyguanosine-3'-phosphoglycolate (dG-3'-PG) prepared synthetically.

Alternatively, to quantitate the nucleosides, the digestion was repeated on 0.2 nmol of 1a (25 µl) as above and subsequently incubated with alkaline phosphatase from calf intestine [specific activity 2000 U/mg, 20 U (1 µl); Boehringer Mannheim] for 30 min at 37°C. The digestion mixture was analyzed by ion pair reverse phase HPLC using an Alltech Adsorbosphere Nucleotide-Nucleoside column (4.6 x 250 mm) and a linear gradient of 0–50% B over 20 min and of 50–90% B from 20 to 30 min (flow rate 1.0 ml/min). Buffer A contained 60 mM KH2PO4, 5 mM tetrabutylammonium bromide; buffer B contained 5 mM tetrabutylammonium bromide in methanol. The peaks from the resulting HPLC trace were quantified by comparison to known amounts of nucleoside standards including dG-3'-PG. Compound, retention time, amount observed, amount calculated (1a): dC, 7.0 min, 1.29 nmol, 1.4 nmol; dG, 10.4 min, 0.75 nmol, 0.8 nmol; T, 11.1 min, 0.91 nmol, 1.0 nmol; dA, 13.1 min, 1.1 nmol, 1.0 nmol; dG-3'-PG, 19.5 min, 0.15 nmol, 0.2 nmol; dG-3'-phosphohexakis(ethylene glycol), 20.8 min, 0.3 nmol, 0.4 nmol. Compound, retention time, amount observed, amount calculated (1b): dC, 7.8 min, 2.63 nmol, 2.64 nmol; dG, 11.1 min, 1.96 nmol, 1.98 nmol; T, 11.7 min, 1.34 nmol, 1.32 nmol; dA, 13.3 min, 1.21 nmol, 1.32 nmol; dG-3'-PG, 19.8 min, 0.24 nmol, 0.33 nmol; dG-3'-phosphohexakis (ethylene glycol), 20.6 min, 0.6 nmol, 0.66 nmol.

NMR sample preparation
1a was desalted using a microdialysis chamber and 2.5 mM NaH2PO4 (pH 6.9) for 24 h. The solution was concentrated in vacuo and lyophilized several times from D2O (99.9%; Cambridge Isotope Laboratories). Compound 1a was dissolved in 0.6 ml of D2O (99.999%; Isotec) and annealed at 65°C, giving ~1.9 mM duplex 1a in 40 mM phosphate buffer (pH 6.8). Compound 1b was 1.4 mM.

NMR experiments
All NMR experiments were performed on custom-built 500, 600 or 750 MHz NMR spectrometers at the Francis Bitter Magnet Laboratory. The acquired data were transferred to a Silicon Graphics workstation and processed using Felix software, version 95 (Acclerys Inc.). 1H and 13C chemical shifts were referenced to an internal standard, sodium 3-(trimethylsilyl)-1-propanesulfonate (TSP) at 0 p.p.m.

PECOSY (flip angle mixing pulse of 36°), TOCSY (30, 60, 100, 150 and 250 ms mixing times) and NOESY (100, 200, 250 and 400 ms mixing times) experiments were recorded at 750 MHz at 20°C in D2O or at 4°C in 90% H2O, 10% D2O. Data sets of 4096 x 512 complex points were acquired with spectral widths of 8000 Hz in both dimensions and 32 scans per t1 increment. During the relaxation delay period, a presaturation pulse of 2 s was used for solvent suppression. For the NOESY experiments in 90% H2O/10% D2O, a Watergate gradient pulse sequence (34) was used for water suppression. For Watergate experiments data sets with 4096 x 512 complex points were acquired with spectral widths of 15 000 Hz in both dimensions. For all experiments, spectra were zero filled to 4096 points in the t1 dimension. The data were processed with a combination of exponential and Gaussian weighting functions. Baselines were corrected with a polynomial or an automatic baseline correction routine in t2 when necessary.

1H-31P GE-HSQC experiments (35) were carried out at 20°C and at 500 or 600 MHz. Data sets contained 1024 x 256 complex points with a spectral width of 6000 Hz in the proton channel and 600, 2000 and 5000 Hz in the 31P channel. Experiments were conducted with a typical relaxation delay of 5 s and with d1 delay times of 10 and 20 ms. Sixteen to thirty-two scans for every t1 increment were used.

1H-13C GE-HSQC experiments (36) were acquired at 600 MHz at 20°C with 4096 x 512 complex points and a spectral width of 5000 Hz in the 1H dimension and 30 000 Hz in the 13C dimension. Transients (128) were recorded for each t1 increment. Long range 1H-13C couplings were detected by choosing dephasing–phasing delay times ({tau}) of 30 and 40 ms (37). BASHD-TOCSY (38) experiments were recorded with a mixing time of 80 ms and 128 transients. Data sets were collected at 4096 x 64 complex points with spectral widths of 100, 200 and 400 Hz for scaling factors of 3, 2 and 1 in the second dimension and 6000 Hz in the first dimension. All 31P spectra were referenced indirectly through the gyromagnetic ratio to trimethyl phosphate by external calibration on TSP (39).

Molecular modeling
Distance constraints. Distance constraints were derived from a 200 ms NOESY experiment at 750 MHz. Peak volumes were assigned by visual inspection using H2'-H2'' and/or cytosine H5-H6 NOEs as a guide for strong NOEs. NOEs were classified as strong, medium or weak with distances of 1.7–4.0, 2.0–5.0 and 3.0–6.0 Å, respectively. NOE volumes were also calculated with the peak pick protocol in Felix 95, and the measured volumes agreed well with those derived from visual inspection. Hydrogen bonds between base pairs were included as restraints for all of the residues. Experimentally determined NOEs (548) were included in the molecular dynamics calculations.

Dihedral angle constraints. Coupling constants for H1'-H2' and H1'-H2'' were derived from PECOSY experiments. Dihedral angle constraints consistent with a C2' endo conformation were used for all residues for which coupling constant information was available. For these residues the dihedral angle H1'-C1'-C2'-H2'' was set at 30 ± 25° and the dihedral angle H1'-C1'-C2'-H2' was set at 150 ± 25°. Coupling constants were measured for H3'-31P with a BASHD-TOCSY experiment (38). The H3'-31P coupling constants were used to calculate dihedral angles using standard methods (40). Only the H3'-31P angle corresponding most closely to the one found in B-form DNA [nH3'-nC3'-nO3'-(n + 1)P between –68° and –98 ± 25°] was used in the modeling protocols. Backbone dihedral angles not determined experimentally were set for nH3'-nC3'-nO3'-(n + 1)P at –80 ± 30°. C4'-C5'-O5-P dihedral angles for all residues were set at –145 ± 30°, except for residue 8, which was not constrained. A total of 95 dihedral constraints were included in the molecular dynamics calculations.

Building of initial coordinate files. Compounds 1a and 1b were initially constructed in InsightII version 95 (Acclerys Inc.) as B-form duplex DNA by removal of the base and C1' to C3' and replacement of C4' with a carboxylate at the site of the PG lesion. The hexaethylene glycol linker was not included in the modeling files. Modifications to allow for a PG in the molecular dynamics calculations were also added in the parameter and topology files of the CHARMm force field used for all subsequent calculations (41). In these files the base and the sugar C1' through C3' were removed from a nucleotide. A carboxylate replaced C4' (CHOH). The PG C4' and C5' and their substituents were modified with appropriate bonds, angles and electrostatic charges based on an aspartate residue (42). The initial atom positions for 1a built in InsightII were read into XPLOR 3.851 on a Silicon Graphics (SGI) workstation. Using the CHARMm force field, the molecular structure file (psf) and the molecular coordinate file (pdb) necessary for the molecular dynamics calculations were constructed.

Molecular dynamics calculations in vacuo. All experimental constraints were included in initial minimizations, molecular dynamics trajectories and final minimizations. Initial coordinates for 1a were energy minimized at 300 K with 200 steps of conjugate gradient minimization. Molecular dynamics trajectories were then run for 100 ps at 300 K. Throughout the energy minimizations and molecular dynamics trajectories, planarity constraints were included for the purine bases at 50 kcal mol–1 Å–2. The NOE force constant was 50 kcal mol–1 Å–2, while the dihedral angle force constant was 40 kcal mol–1 rad–2. These force constants were chosen to minimize NOE distance constraint and dihedral angle constraint violations. Non-bonded interactions had a cut-off of 11.5 Å. The non-bonded interaction was switched from on to off between 9.5 and 10.5 Å. During the molecular dynamics calculations, atomic coordinates were written to a trajectory file every 0.2 ps throughout the 100 ps molecular dynamics trajectory. The structure was averaged over the last 10 ps of the run, and the averaged structure was again subjected to 200 steps of conjugate gradient minimization. Ten averaged structures were calculated. Coordinates of the 10 averaged structures of 1a and 1b have been deposited in PDB. The coordinates for 1a are in PDB 1G5K and 1GJ1 (10 structures and one averaged minimized structure, respectively). The coordiates for 1b are deposited in PDB 1N0O and 1N0K (10 structures and one averaged minimized structure, respectively).

Back-calculations of NMR spectra. Back-calculations were performed in InsightII using the iterative relaxation matrix (IRMA) approach (43,44). The entire 200 ms NOESY spectrum at 750 MHz was back-calculated for the average of 10 structures of 1a where each structure resulted from a separate 100 ps molecular dynamics run. The spectra were calculated using the full relaxation matrix. Parameters for the IRMA calculations were set as follows: the rotational correlation time ({tau}c) was 3 ns and T1 leakage was 10 s–1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Choice of the sequences
The sequence of the duplex 1a containing a PG lesion is shown in Figure 2A. Design considerations of 1a encompassed having a full turn of a DNA helix and a single BLM binding site (5'-GTAC-3'), located opposite the lesion. This context was chosen because we have previously demonstrated that 5'-GTAC-3' is a ‘hot spot’ for the generation of ds DNA breaks by a single BLM (6). This lesion in the presence of a metallo-BLM has allowed us to test our model for BLM reorganization during the ds cleavage process (7). Our initial efforts focused on the synthesis of 1a without crosslinking the ends. This construct led to a highly mobile duplex (Fig. 4A). Only seven of the possible 12 imino protons of this three piece complex were clearly visible at 4°C. To improve the thermal stability of the duplex, the two strands were crosslinked with the non-nucleotide, polyethylene glycol linker (45). Previous work by Altmann et al. and more recent work on a nicked DNA duplex (46) have shown that hexaethylene glycol linkers are very effective in increasing duplex stability, without distortion of their structure (45). 1a was therefore synthesized (see below) and the 1D 1H NMR (90% H2O/10% D2O) was recorded (Fig. 4B) at 4°C. Twelve imino signals are apparent, two of which are very broad. Thus we have adopted the hexaethylene glycol spacer methodology so that we could obtain a structural model of this lesion. The structure of the lesion complexed to HOO-CoBLM has been previously reported (29).



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  		Figure 4. Imino region in the 1D Watergate NMR (750 MHz) experiment (90% H2O/10% D2O) at 5°C. (A) 1a without hexaethylene glycol linkers. (B) 1a (G6 and T19 are putative assignments). (C) 1b (G6 chemical shift of 10.5 p.p.m. and G19 are putative assignments).

 
Compound 1b (Fig. 2C), which contains a hot spot for BLM-mediated ss cleavage, was chosen as a control sequence unable to undergo BLM-mediated ds cleavage. In addition, the structure of this lesioned DNA would serve to address the importance of sequence context on the structures of gapped 3'-PG/5'-P lesions and their recognition by repair enzymes.

Solid support DNA synthesis
A prerequisite for structural NMR studies of PG containing lesions is the development of a reliable synthetic strategy for their large-scale synthesis using phosphoramidite chemistry. The design of the oligonucleotide containing the lesion requires the introduction of three modifications into the standard solid phase DNA synthesis protocol to synthesize the lesion precursor 5 (Fig. 3). Previous studies have developed a modified controlled pore glass solid support, allowing the incorporation of a glycerol unit at the 3' end of the oligonucleotide (Fig. 3, 2) (32). They have further demonstrated that after deprotection, the precursor can be oxidized under mild conditions to the PG by solution phase chemistry (33). As noted above, incorporation of hexaethylene glycol phosphoramidite building blocks (Fig. 3, 3) allows the crosslinking of both DNA strand ends. The final step of the solid phase synthesis is the attachment of a 5'-phosphate precurser using 4 (Fig. 3) that leads to 5 (47). The length of the sequence and the incorporation of the three different modifications made the solid phase synthesis challenging. Initial attempts to synthesize 5a by an unmodified Applied Biosystems programmed DNA synthesis cycle resulted in the occurrence of failure sequences and a low overall yield. In particular, the coupling efficiency of the linkers varied from 80 to 95%. Therefore, a longer coupling time for the spacer was chosen, which improved the yield to 90–96%. Decreased coupling yields (<95%) were also observed for the last steps of the synthesis. Initially, longer reaction times (2–10x) and excess reagents (2x) were tried to enhance coupling efficiency. Evaluation of the crude deprotected oligonucleotides by ion exchange HPLC showed ultimately that increased amounts of side products resulted from acid-catalyzed depurination by the tetrazole activator. A significant improvement in the purity of the product was thus achieved by changing tetrazole to dicyanoimidazole. The advantages of this reagent are its reduced acidity (pKa 5.2) and its increased reactivity in comparison to tetrazole.

In the final step of the solid phase synthesis, the chemical phosphorylation reagent (4) was coupled to the oligonucleotide by omitting the capping steps in the programmed synthesis cycle and by using a coupling time of 6 min. The lesion precusor was then cleaved from the column and deprotected by standard methods (Fig. 3, step a). The presence of a dimethoxytrityl (DMT) group at the 5'-P end of the lesion allowed an efficient separation of the lesion precursor from failure sequences by reverse phase HPLC. In a final step, the 5'-P was deprotected by removal of the DMT group with acetic acid and subsequent ammonolysis (Fig. 3, step b). The isolated yield of 6a was ~23%.

Oxidation of 6
Compound 6 was converted to 1a by a two-step oxidation procedure (Fig. 3, steps c and d) (32,33). First, the terminal diol of the 3' glycerol was cleaved with sodium periodate to generate the 3' glycol aldehyde 7 (>95% conversion). Second, 7 was oxidized to the acid using sodium chlorite. The different retention times of 6, 7 and 1 on chromatography using an ion exchange HPLC system made it possible to monitor the course of the reaction (Supplementary Material, Fig. S1). The crude oxidation product (~75% purity by HPLC) was purified further by semi-preparative ion exchange HPLC to yield ~1.2 µmol (54%) of 1a. The purity of 1a was >95%, based on ion exchange and reverse phase HPLC chromatograms. The identity of the oligonucleotide was confirmed by ES-MS. Compound 1b was generated in similar overall yield and characterized by both HPLC and ES-MS.

Quantitation of the PG by enzymatic digestion
To characterize 1a further, an enzymatic digestion scheme was developed. This procedure allowed the detection and quantitation of the dG-3'-PG. 1a was digested with two enzymes: DNase and SVPDE. DNase cuts DNA randomly into small pieces with an average size of 3 nt. The resulting cleavage products are phosphorylated at the 5' end and contain free 3'-OH groups. SVPDE further digests these oligonucleotides into 5'-nucleotides. Nucleotides with a 3' modification like the PG or the hexaethylene glycol are not substrates for this enzyme under the chosen reaction conditions. The 3' modified nucleotides were separated from the 5'-nucleotides by ion exchange HPLC. Two peaks (A and B) in the chromatogram had significantly longer retention times than the 5'-nucleotides. Peak A (retention time 5.5 min) was desalted by reverse phase HPLC and analyzed by ES-MS. The molecular mass of 691.8 g/mol is in agreement with the chemical structure of 5'-phosphodeoxyguanosine-3'-phosphohexakis(ethylene glycol). Peak B (retention time 9.2 min) co-eluted with the synthesized reference compound, dG-3'-PG, and had an identical UV/vis spectrum.

To quantitate the amount of dG-3'-PG relative to the other nucleotides, the entire reaction mixture subsequent to treatment with DNase and SVPDE was incubated with alkaline phosphatase. This enzyme hydrolyzes the 5'-monophosphates, leaving nucleosides that were then characterized by C18 reverse phase ion pairing HPLC. The resulting chromatogram (Supplementary Material, Fig. S2) showed baseline resolution of the deoxynucleosides (dC, dG, T and dA), the dG-3'-PG and dG-3'-hexakis(ethylene glycol). The amounts of the nucleosides were compared to a reference mixture containing their expected ratios based on the sequence. The amount of all nucleosides was in agreement with the predicted structure; the dG-3'-PG accounted for 75% of the expected value.

Assignment of the proton chemical shifts
The base and sugar protons of 1a (and 1b) were assigned by standard 2D NMR methods (48). The sugar spin systems were identified by through bond correlations, measured by TOCSY experiments with mixing times of 60 and 90 ms. The 5' and 5'' hydrogens were assigned based on TOCSY (150 ms mixing time) and NOESY (250 ms mixing time) experiments. Table S1 (Supplementary Material) contains the chemical shifts of most of the protons of 1a. Table S2 (Supplementary Material) contains the chemical shifts of most of the protons of 1b.

The 250 ms NOESY experiment at 20°C was used for the detection of sequential NOE connectivities. The observed NOE intensities are typical for B-form DNA and can be attributed to a single conformation of 1a in solution. Sequential NOE connectivities can also be traced in the lesion containing strand from A1 to G6 and from A8 to G13 (Fig. 5A). In 1a it was not possible to detect any NOE interactions between G6 and A8, suggesting that there is no collapse of the gap between these two bases. The NOESY data allow a description of the structural features of the nucleotides flanking the lesion as well. Sequential NOEs between the sugar hydrogens (H1', H2' and H2'') of A5 and H8 of G6 indicate that the 3' PG modification of G6 does not significantly alter the stacking of G6 with A5. For example, a strong NOE is detected between A5 H1' and G6 H8 (Fig. 5A, arrow A). The only structural perturbation of B-form DNA on this side of the lesion is apparent from an unusually strong interstrand NOE between A5 H2 and T23 H1' (Fig. 5B, arrow D).



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  		Figure 5. Base to H1' region of the 250 ms NOESY spectrum at 20°C (750 MHz). (A) Solid lines indicate the NOE connetivities from C1 to G6 (black) and from A8 to G13 (red). The following NOE cross-peaks are labeled by an arrow: A, A5 H1' to G6 H8; B, A8 H1' to C9 H6; C, A8 H8 to C9 H5. (B) Solid lines indicate the NOE connetivities from A17 to T24. Red lines indicate the NOE connectivities from the residues T19 to C21 that are located opposite the lesion. The following NOE cross-peaks are labeled: A, T19 H1' to A20 H8; B, A20 H1' to C21 H6; C, A20 H8 to C21 H5; D, A5 H2 to T23 H1'.

 
The influence of the lesion is also apparent at the 5'-P side. A8 H8 is significantly downfield shifted (~0.3 p.p.m.), outside the envelope of the base protons (Fig. 5A). Despite this perturbation, NOEs between A8 H1' and C9 H6 (Fig. 5A, arrow B) and between A8 H8 and C9 H5 (Fig. 5A, arrow C) indicate stacking interactions between these residues.

NOE connectivities can also be traced along the intact strand from C14 to G26. Figure 5B shows a portion of the base to H1' connectivities from A17 to G25. The NOESY data indicate that there are also minimal structural perturbations of the residues in the intact strand opposite to the lesion. There are no interruptions of the sequential NOE connectivities from G18 to C21 (Fig. 5B). NOE cross-peaks between A20 H1' and C21 H6 (Fig. 5B, arrow B) and between A20 H8 and C21 H5 (Fig. 5B, arrow C) are indicative of stacking interactions between the adenine A20 and the cytosine C21. The only apparent perturbation is the decreased intensity of T19 H1' and A20 H8 (Fig. 5B, arrow A).

Assignment of the exchangable protons (49)
The Watergate NOESY data at 5°C show 12 distinct signals in the imino region from 12–14 p.p.m. (Fig. 4B) and 10 of these 12 protons show NOE interactions allowing chemical shift assignments. The imino protons belonging to base pairs G6 C21 and A8 T19 broadened in the 1D spectra (Fig. 4B) and are not detectable in the 2D spectra. The broad signal at 12.8 p.p.m. has been assigned to the G6 imino proton based on our ability to assign hydrogen bonding of the amino protons of C21 within this base pair. The two amino protons (7.86 and 6.99, Supplementary Material, Table S1) have been assigned based on their NOEs to their own C21 H5. However, NOEs between these protons are only one third of the intensity of the NOEs between the amino groups of the other cytosines, indicative of exchange. Furthermore, the chemical shift of the hydrogen bonded proton is upfield shifted 0.5–0.7 p.p.m. relative to the unperturbed base pairs, also indicative of enhanced exchange. Increasing the temperature of the sample resulted in duplex melting from the lesion towards the linkers. At 25°C the imino protons of the base pairs G18 C9 and A5 T23 showed significantly reduced peak intensities in comparison to the signals of the other imino protons that are closer to the linker.

1H-31P NMR GE-HSQC measurements
31P chemical shifts of the DNA phosphodiesters can be a sensitive indicator of an altered DNA conformation (50,51). 1H-31P GE-HSQC experiments were therefore undertaken and revealed a narrow dispersion of signals (–2.4 to –3.3 p.p.m.) including that associated with the 3'-PG, ruling out significant structural backbone perturbations of the intact strand and of the 3'-PG. Altered 31P chemical shifts are, however, associated with the linker connections at the 5'-ends of both strands of 1a and the 5'-P end at A8 (45). The chemical shifts of the phosphates associated with the linkers are downfield shifted by ~1 p.p.m. relative to generic phosphodiesters, in agreement with the literature (45). The 5'-P end at A8 of 1a is observed at ~0.3 p.p.m., typical for monophosphates (25). This assignment is based on a cross-peak of the H4' of A8 and this P. Detection of this 5'-P, due to its altered relaxation properties, required a change in the delay time ({tau}a) from 10 to 20 ms in the GE-HSQC experiment (Supplementary Material, Fig. S3).

Assignment of the PG protons
Assignment of the methylene protons of the PG lesion is required to detect interactions with its environment. A 13C-HSQC experiment was used to make a preliminary assignment of the PG protons and their carbon chemical shift to 4.23 and 67.5 p.p.m., respectively. The low spectral dispersion of the H5'/H5'' deoxyribose protons prevented unambiguous assignment of PG protons and therefore a long range GE-HSQC experiment was used as further confirmation. This experiment involves polarization transfer through three bonds from the methylene protons of the PG to its carboxylate carbon. A cross-peak was observed between protons at 4.21 p.p.m. and the carboxylate at 178.9 p.p.m. (Fig. 6A). The proton chemical shift agrees with the assignment made using the HSQC experiment and published values for model compounds containing a 3'-PG (52,53). For a comparison of signal intensities the coupling between the methyl groups of the thymine and the C6, C5 and C4 carbons of the pyrimidine ring are also shown (Fig. 6B). The assignment of the methylene protons of the PG allowed careful examination of the NOESY spectra to look for interactions with other residues of the DNA. Unfortunately, all of the NOEs could be assigned to interactions between 5'/5'' protons and other hydrogens. The lack of detectable interactions indicates flexibility in its conformation.



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  		Figure 6. A long range GE-HSQC experiment. (A) A cross-peak between the carboxylate of the phosphoglycolate moiety at 178.9 p.p.m. and the protons of the phosphoglycolate at 4.21 p.p.m. (B) Cross-peaks between the methyls of the thymines in 1a and the C5 (113 p.p.m.), C6 (139 p.p.m.) and C4 (169 p.p.m.) carbons of the pyrimidines.

 
Sugar and backbone conformations
The PECOSY data allowed the determination of most (41/52) of the H1' to H2'/H2'' coupling constants of the deoxyriboses of 1a (Supplementary Material, Table S3). The values for J1',2' were between 7.8 and 9.8 Hz, indicative of an S-type sugar conformation and the 2' endo sugar pucker found in B-form DNA. The coupling constants of the nucleotides A8 (J1',2' = 7.8 Hz and J1',2'' = 5.9 Hz) and G6 (J1',2' = 9.8 Hz and J1',2'' = 5.8 Hz) flanking the lesion are also consistent with a B-like conformation. Finally, the phosphodiester backbone conformation was determined by BASHD-TOCSY experiments. These measurements allowed the calculation of 17 dihedral angles ({epsilon}) between the 3'-hydrogens and their corresponding phosphates, giving rise to {epsilon} values between 150° and 172° (Supplementary Material, Table S4). Thus these measurements support the results from the 31P chemical shifts that there are no significant structural perturbations of the DNA backbone. The only exception is the dihedral angle associated with the P linked to the glycolate in which the value of {epsilon} is 142°.

Molecular dynamics runs
The molecular modeling used 548 experimentally determined distance constraints and dihedral angle constraints for the backbone and the sugar conformations. Since the presence of the hexaethylene glycol spacers allowed assignment of all 12 imino protons, hydrogen bonds between base pairs were constrained for all residues, including the ones at the oligonucleotide ends. Ten separate molecular dynamics runs starting from B-form DNA were performed to give 10 separate structures. The overlaid structures resulting from these runs (Fig. 7) show good agreement. The nucleotide opposite to and the nucleotides adjacent to the PG lesion exhibit a regular B-form conformation. The PG is extrahelical in all 10 structures. The 3'-PG and the 5'-P at A8 are disordered since there are no distance constraints limiting their position. Their flexibility is shown in Figure 8. The distance between the 5'-P and the 3'-PG phosphate is 8 Å, in comparison with a value of 6.5 Å for B-form DNA. Electrostatic repulsion between the negatively charged PG and the 5'-P may be the cause of this increased distance. Table 1 summarizes the results of the molecular modeling.



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  		Figure 7. Stereo view of 10 structures of 1a. The hexaethylene glycol spacers are not shown because they were not included in the modeling. The 3'-PG lesion is indicated by an arrow.

 


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  		Figure 8. Conformational disorder in the 3'-PG and the 5'-P ends of 1a. Four out of 10 structures from Figure 6 are shown. Each structure is shown in a different color. The average of 10 structures is shown for the strand opposite the lesion (T19–C21, in black).

 

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  		Table 1. Summary of molecular modeling of 1a
 
Back-calculating structures
The entire 200 ms NOESY spectrum for 1a was back-calculated using the IRMA protocol in InsightII. An ensemble of 10 structures was back-calculated (Supplementary Material, Fig. S4). All of the experimentally observed peaks are observed in the back-calculated spectrum with intensities similar to those observed experimentally.

Titration of 1b with HOO-CoBLM
The structure of 1b was obtained from 762 NOE-derived distance restraints and 47 dihedral angle restraints [15 from BASHD-TOCSY experiments (H3'-31P) and 22 from PE-COSY] using the protocols described for 1a. The resulting structural model is very similar to 1a (Supplementary Material, Fig. S5).


   DISCUSSION
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY MATERIAL
 REFERENCES
 
The long range goals of our structural studies on 1a and 1b are to understand the mechanism of BLM-mediated ds cleavage and to understand how both ss and ds DNA lesions containing PG moieties can be recognized and repaired. Since the GTAC sequence is a hot spot for ds cleavage (6) and the GGCC sequence is a hot spot for ss cleavage with little ds cleavage, a 3'-PG lesion next to a 5'-P was studied in each sequence context. It was hoped that a comparison of the structural models for these two lesions might provide insight into both processes.

Information from 2D NMR spectroscopy has been used to provide the first structural models of gapped lesions containing a 3'-PG and a 5'-P end (Fig. 7 and Supplementary Material, Fig. S5). All of the modeled structures for 1a and 1b show the PG as extrahelical and the 5'-P disordered with the average distance between two phosphates adjacent to the gap greater than in B-form DNA. Furthermore, the 1D NMR of the oligomers (Fig. 4) in water indicate that the imino protons adjacent to the gap exchange more rapidly than the remaining imino protons in the duplexes, suggesting flexibility. Both duplex structures are B-form with the base opposite the gap and bases adjacent to the gap well stacked.

The similarities in structure between 1a and 1b by themselves provide little insight into why the intact duplex of 1a provides an excellent ds cleavage site for BLM while the duplex of 1b does not. Our previous studies have shown that titration of 1a with HOO-CoBLM led to a single complex whose structure was readily determined (29). A similar titration of 1b with HOO-CoBLM was carried out in this study in an effort to understand the basis for the difference in behavior of these two oligomers with respect to ds cleavage. The results with 1b were not easily interpretable. Multiple binding modes were apparent precluding detailed chemical shift assignments (data not shown). The results of the 1b titrations are similar to those observed by titration of lesioned GXAC where X is an abasic site (instead of the 3'-PG/5'-P of 1a) with HOO-CoBLM. The abasic site lesion does not lead to ds cleavage and the titration with HOO-CoBLM also leads to multiple complexes (29). Our structural model of 1a complexed with HOO-CoBLM (29) suggests that one excellent ss cleavage site appropriately spaced on each strand of duplex DNA is essential for efficient ds cleavage. However, both the nature of the sequence context and the nature of the lesion appear to be important in BLM-mediated ds cleavage in ways not yet understood.

Despite the prevalence of gapped DNA duplexes generated by numerous DNA damaging processes little information is available about their structures and the basis of their recognition by DNA repair enzymes. Recent NMR studies and molecular dynamics simulations have focused on gapped duplexes with 3' and 5' OH ends opposite guanine and adenine (54,55). In both cases the NMR data revealed a single major species in which the DNA was B-like and the A and G opposite the gap well stacked in the helix. The results from molecular dynamics simulations indicated in both cases several families of structures, with straight and bent DNA conformations. These studies indicate, as do ours on 1a and 1b, that duplex DNA is, on average, unperturbed from B-form, but flexible in the region of the gap.

Recent structures of two types of endonucleases, APE1 and Endo IV, in complex with the same THF (an analog of an abasic site)-containing oligonucleotide support the importance of flexibility in recognition of substrate and provide insight into additional factors that might influence the recognition process (14,28). In both structures the deoxyribose analog is flipped out of the helix and into a specific binding pocket on the enzyme (14,28,56). In both cases the DNA is bent (35° and 90°, respectively) away from the lesion and in the case of APE1 a comparison between the C{alpha} backbone of free and oligonucleotide-bound structure reveal a r.m.s.d. of ~0.7 Å. These data support the protein’s ability to recognize flexibility in the substrate and suggest the importance of a protrusion from the duplex phosphodiester backbone in the recognition process. In both structures the size of the pocket that accomodates the lesion is small.

While there are no structures of oligonucleotides containing gapped 3'-PG, 5'-P lesions bound to repair enzymes, both APE1 and Endo IV can repair this lesion. Thus, as suggested by the THF-APE1 structure, both flexibility and the 3'-PG protrusion from the helix in 1a and 1b may be important in the recognition of this type of lesion by these repair enzymes. The PG lesion is also small enough to fit into the enzyme’s binding pocket (14), as can 3'-phosphate and 3'-phosphoglycoaldehyde lesions also repaired by these enzymes (15,57). Thus the charge of the PG does not appear to be crucial for recognition by APE1. Nevertheless, in vitro the recognition and repair of abasic sites appears to be more efficient than the recognition of PG lesions (11,12). It is unclear at this point what elements contribute to the lower repair efficiency and if this decreased efficiency is also observed in vivo. The detailed mechanism of repair of different lesions and which step(s) is rate limiting remain to be established.

In addition to substrate flexibility and extrahelicity, APE1 appears to recognize a space bracketed by duplex DNA, suggested by its ability to bind tightly to DNA containing a single-nucleotide gap with a 3'-OH end and a 5'-P end, but not to nicked DNA with the same ends (12). This putative space bracketed by duplex DNA is seen in our structural model for the PG lesion and is thus consistent with this indicated requirement for recognition (Fig. 6).

Many of the studies on the repair of PG lesions have been carried out on oligonucleotide substrates. However, PG lesions have also been ligated into specific vectors to test ss and ds break repair in cell extracts and intact cells (58–60). When APE1 is neutralized by polyclonal antibodies, repair of ionizing-radiation induced ss damage such as 3'-PG ends still occurs in partially purified HeLa cell nuclear extracts (60). Thus under these conditions other phosphodiesterases are responsible for the repair of these lesions. Nevertheless, when the amount of APE1 is reduced in vivo by APE1 antisense RNA expression, hypersensitivity of the cells results with respect to oxidizing agents that can generate PG lesions (61,62). Furthermore, recent studies with cells derived from germ cell tumors with overexpressed APE1 suggest that the cells are more resistant to BLM, providing further support that this enzyme plays an important role in repairing the damage created by the drug (17). As indicated in Figure 1, however, the 4'-ketoabasic site similar to the abasic site is also produced by BLM and might be the predominant lesion repaired by APE1.

The details of PG lesion repair in vivo remain to be elucidated, and the importance of understanding the structure of this lesion is evident. The ds DNA breaks induced by BLM all contain PG lesions, and PG lesions at ds DNA breaks are poor substrates for APE1 (12), the only human phosphodiesterase capable of removing this lesion characterized to date (11–13,18,20,21). The ability of this protein to interact with other proteins in the BER repairosome could, however, alter the rate of repair in ways that remain to be determined. The cytotoxicity of BLM is thought to be related to its ability to cause ds DNA breaks (63). Thus understanding the structure of a PG lesion is an important step toward understanding the BLM-induced ds cleavage mechanism (29) and the mechanism of repair of the lesions generated by the drug.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at NAR Online.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH grant GM34454 to J.S. The NMR facility is supported by NIH grant RR-00995.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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