Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hang, B.
Right arrow Articles by Singer, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hang, B.
Right arrow Articles by Singer, B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 273, Issue 50, 33406-33413, December 11, 1998


Correlation between Sequence-dependent Glycosylase Repair and the Thermal Stability of Oligonucleotide Duplexes Containing 1,N6-Ethenoadenine*

B. Hang, J. Sági, and B. SingerDagger

From the Donner Laboratory, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Previous experiments on DNA sequence context reported that base modification, replication, and repair are affected by the nature of neighbor bases. We now report that repair by mammalian alkylpurine-DNA-N-glycosylases (APNG) of 15-mer oligonucleotides with a central 1,N6-ethenoadenine (epsilon A), flanked by 5' and 3' tandem bases, is also highly sequence dependent. Oligonucleotides with the central sequences -GGepsilon AGG- or -CCepsilon ACC- are repaired 3-5-fold more efficiently than those containing -AAepsilon AAA- or -TTepsilon ATT- when using human or mouse APNG. Melting curves of the same duplexes showed that oligomers with G·C/C·G neighbors were less denatured than those with A·T/T·A neighbors at 37 °C. This sequence-dependent difference in denaturation correlates with the relative thermodynamic stability of oligomers with G·C/C·G or A·T/T·A neighbors. The dependence of repair on thermal stability was confirmed by enzyme reactions performed over 0-45 °C. Under these conditions, repair of epsilon A flanked by G·C/C·G was dramatically increased at 37 °C with continuous increase up to 45 °C, in contrast to that with flanking A·T/T·A pairs, which was in agreement with the degree of denaturation of these duplexes. These results indicate that the thermodynamic stability conferred by base pairs flanking epsilon A plays an essential role in maintaining the integrity of the duplex structure which is necessary for repair.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Many laboratories have published data on the effect on efficiency of modification, replication, or repair of nucleic acids conferred by the immediate neighbor bases. In almost all cases, there was some measurable difference depending on sequence context. Most of the experiments on chemical modification which used a gene or a naturally occurring DNA led to a statistical analysis of the bases neighboring the lesion studied. These important data contributed to the hypothesis that localized DNA structure was an important determinant in the non-random distribution of adducts (e.g. Refs. 1-10).

Replication efficiency has been found in many cases to be a function of nearest neighbors (e.g. Refs. 11-21). In these experiments, specific sequences containing a modified base were primed and insertion and extension examined using a variety of polymerases. Usually the 5' neighbor base effect was found to be an important factor, although the 3' neighbor can also play a role. In addition, it has also been reported that the proofreading efficiency of polymerases is sequence-dependent (11, 17).

There are also numerous studies on the sequence dependence of enzymatic repair of various types of damage in defined DNA sequences (e.g. Refs. 22-32). Here again, specific neighbor sequences led to significant variations in the rate and extent of excision of a modified base by repair mechanisms including base excision repair, nucleotide excision repair, mismatch repair, and O6-methylguanine-DNA methyltransferase.

When a double-stranded structure is required, either as a partial duplex (template/primer) or as a full-length duplex, the stability of base pairs adjacent to the adduct can be an important factor in replication and repair. Thermodynamics has been a general method used to probe nucleic acid structure and strandedness (e.g. Refs. 33-38). In some replication studies of templates containing modified bases, a correlation between the rate of insertion of a dNTP and the relative base pairing energies of the nearest neighbor base pairs was reported (16, 39, 40).

In repair, it was assumed that G·C-rich neighbor regions would play a role in stabilizing the necessary double strand containing a mismatched base (22). This is likely to be true for all DNA modifying and repair enzymes which require a DNA duplex structure for activity except uracil-DNA glycosylase (41) and O6-methylguanine-DNA methyltransferase both of which can act on single-stranded substrates, although O6-methylguanine-DNA methyltransferase is more active on double-stranded DNA substrates (42, 43). However, under the circumstances where repair is carried out by protein complexes such as the Escherichia coli UvrABC nuclease, the mechanism(s) underlying the sequence-dependent repair becomes more complicated as the efficiency of repair is also correlated with the stability of the preincision UvrB·DNA and UvrBC·DNA complexes (30, 31).

In the present work, we have carried out parallel studies of both repair by alkylpurine-DNA-N-glycosylase (APNG)1 (also termed 3-methyladenine-DNA glycosylase) and thermal stability using a set of specifically designed 15-mer oligonucleotides with purine or pyrimidine 5' and 3' tandem flanking bases to a central 1,N6-ethenoadenine (epsilon A). This adduct is efficiently removed by mammalian APNGs (44-46), which, in common with most repair enzymes, require a double-stranded duplex for activity (47). We have now found a connection between the rate and extent of the APNG-mediated cleavage of epsilon A-containing 15-mers and the thermal stability of these duplexes. Our work appears to be the first correlation between sequence-dependent glycosylase repair and degree of denaturation of the oligomer substrates. This may provide insights into sequence specificity of in vivo repair of epsilon A or other adducts requiring a stable duplex structure. The present work uses epsilon A as a representative mutagenic adduct (48-50) which has been shown to be produced both exogenously and endogenously (51).

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- HeLa cells were obtained from Cell Culture Center, Endotronics Inc., Minneapolis (MN), a National Institutes of Health sponsored facility. The [gamma -32P]ATP (specific activity 6000 Ci/mmol) was purchased from Amersham. T4 polynucleotide kinase was purchased from U. S. Biochemical (Cleveland, OH). 1,N6-Ethenodeoxyadenine phosphoramidite was obtained from Glen Research (Sterling, VA). Formamide, spermidine, bovine serum albumin, 40% acrylamide/bis solution (19:1), and urea were obtained from Sigma. Poly(dI-dC) was purchased from Pharmacia. OPC cartridges were from Applied Biosystems.

Repair Enzymes-- The purified 26-kDa truncated human APNG (0.58 mg/ml) was a gift from Dr. T. R. O'Connor. It was shown to be more heat stable than the full-length human protein (52). In addition, O'Connor reported that there is no significant differences in the kinetic parameters of the full-length human APNG and the truncated form (52). The purified 31-kDa mouse APNG protein (0.8 mg/ml) which lacks 48 residues from the amino terminus of the wild-type (wt) protein without loss of activity (53) was a gift from Drs. R. Roy and S. Mitra. As with the truncated human APNG, there was no difference in kinetic properties of releasing m3A and m7G due to size (53). The purity of both proteins was found to be homogeneous by SDS-polyacrylamide gel electrophoresis analysis. The human 5' AP endonuclease (HAP1) (0.25 mg/ml) was a gift from Dr. I. D. Hickson. The crude HeLa cell-free preparation was used as a source for wild-type APNG protein activity and also an overall measurement of repair in the cell. The preparation of crude extracts from HeLa cells was carried out essentially as described by Singer et al. (44). The ammonium sulfate precipitates, dissolved in a buffer containing 25 mM Hepes-KOH, pH 7.8, 0.5 mM EDTA, 0.125 mM phenylmethylsulfonyl fluoride, 3 mM beta -mercaptoethanol, and 10% glycerol, were used in enzyme assays.

15-mer Oligonucleotides-- The 15-nucleotide long oligodeoxynucleotides with a central A or epsilon A were synthesized on a 1 µM scale and purified as described previously (21). The following 15-mer oligomers were used in this study: 5'-AGCGGNNXNNGAGCT-3', where -NNXNN- are: -GGepsilon AGG-, -CCepsilon ACC-, -AAepsilon AAA-, and -TTepsilon ATT-. All control sequences were the same 15-mers in which A replaced epsilon A in -NNXNN-. The complementary oligodeoxynucleotides synthesized contained a thymine opposite epsilon A or A.

Two complete sequences are shown in the form of duplexes (Fig. 1). The sequences in common are outside the box and are shown as hydrogen bonded (bullet ). The boxed central sequence is presented with unknown strength of hydrogen bonds (open circle ) since it is possible that the region at the epsilon A·T mismatch can be destabilized as reported for normal base mismatches and their neighbor bases (54-56).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1.   Sequences in common (3' and 5' pentamers) and changed sequences (boxed) in two 15-mer oligonucleotides with a central epsilon A and duplex tandem bases. The boxed sequences are shown with unknown strength of hydrogen bonding (open circle ) as a possible effect from the epsilon A·T mismatch.

Enzymatic Reaction-- The 15-mer oligonucleotides were 5'-end radiolabeled and annealed to their complementary strands as described by Rydberg et al. (57). Briefly, the epsilon A- or A- containing oligonucleotides were 5'-end labeled with [gamma -32P]ATP and T4 polynucleotide kinase in a kinase buffer containing 50 mM Hepes-KOH, pH 7.5, 10 mM beta -mercaptoethanol, 10 mM MgCl2 at 37 °C for 35 min. The labeled oligomers were then annealed to complementary 15-mers (1.5-fold molar excess) in a buffer containing 10 mM Hepes-KOH, pH 7.5, 100 mM NaCl by slowly cooling down from 90 °C to room temperature (1 h).

The enzymatic assay used to examine APNG-mediated cleavage of oligonucleotide substrates was carried out essentially as described by Rydberg et al. (57, 58). The standard reaction was performed in a total volume of 10 µl in 35 mM Hepes-KOH, pH 7.8, 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM spermidine, 50 mM KCl, 0.6 mM MgCl2, 400 µg of bovine serum albumin, 10% glycerol with 20 fmol of the 15-mer duplex. In the reactions when crude HeLa extracts were used, 0.5 µg of poly(dI-dC) was added and bovine serum albumin was omitted. The reactions were stopped by addition of equal volume of gel loading buffer containing 90% formamide, 50 mM EDTA, and 0.05% bromphenol blue, heated at 95-100 °C for 2 min. This treatment yielded virtually identical amounts of cleavage of AP sites generated by the action of APNG on epsilon A as that produced by the human 5' AP endonuclease (HAP1) cleavage of the AP sites (Fig. 2). Note that the protein concentration of HAP1 used was greatly in excess (25 ng) over that normally needed to cleave the same amount of AP sites (59). The nonenzymatic method was used for all the cleavage reactions in this work. Electrophoresis of the reaction mixtures was carried out using a 12% polyacrylamide gel containing 8 M urea. The bands corresponding to the cleavage products and the remaining uncut substrates were scanned and quantitated using a Molecular Dynamics PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The curve fitting used the MicroCal Origin program (version 3.0).


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 2.   Comparison of two methods for the cleavage of the AP site in all four epsilon A-containing oligomers resulting from APNG activity. The dotted bars are percent cleavage using 2.5 ng of human APNG while the open bars are using the same experimental conditions except for the use of both 2.5 ng of human APNG and 25 ng of HAP1. Both reactions were 20 min at 37 °C. See "Experimental Procedures" for details.

Effect of Temperature Changes on Cleavage of epsilon A-containing Oligomers by APNG-- For the temperature-dependent experiments the standard enzyme reaction assay described above was used with differing reaction temperatures, 0-45 °C. In one type of experiment, a single reaction mixture was divided into six aliquots and each was incubated at a different temperature for 20 min with 2.5 ng of human APNG. 0 °C indicates on ice, 10 and 20 °C were controlled by adding ice to the water bath to maintain the required temperatures. Samples at 30, 40, and 45 °C were kept in water baths. All incubations were for 20 min with 2.5 ng of human APNG.

In the second experiment, samples under standard conditions with 2.5 ng of human APNG were incubated at 20 °C for the indicated times (20 or 100 min) and then half of each sample continued an additional 20 min at 37 °C. Quantitation of APNG-mediated cleavage was by the standard gel electrophoresis which was scanned by a PhosphorImager as described under "Enzymatic Reaction."

Thermal Melting Experiments-- Molar absorbance of the single strands containing only natural nucleotides was calculated by using the DNA/Oligo Quantitation Software of a Beckman DU 7400 diode-array spectrophotometer. In the case of the epsilon A-containing oligonucleotides, the nearest neighbor interactions (60) were recalculated using the molar absorption coefficient of 5000 for epsilon A, which was calculated from published spectral data (61). Molar absorbance of the epsilon A-containing 15-mers was found to be lower by an average of 6%, as compared with the A-containing corresponding sequences. The double strands were prepared by mixing equimolar amounts of the non-self-complementary single-strands in the buffer containing 0.1 M NaCl, 0.01 M sodium phosphate, and 0.1 mM EDTA, pH 7.0. These conditions differ from those used in enzymatic assays primarily in that the oligomer concentration is higher.

Thermal transition profiles were measured using a Beckman DU 7400 diode-array spectrophotometer at 260 or 280 nm. A buffer containing 0.1 M NaCl, 0.01 M sodium phosphate, and 0.1 mM EDTA, pH 7.0, was used in all thermal denaturation experiments. For the determination of the melting curve at a single duplex concentration or for the analysis of the premelting region of the melting profiles a Beckman special Tm-6-cell holder was used. This has an internal thermometer, a Peltier temperature controller and is equipped with gas inlet accessory. The latter was used to flush the cell holder with nitrogen when measurements were started below 16 °C. The path length was 1 cm and the volume of samples was 0.32 ml. For the concentration dependence measurements, the Beckman normal 6-cell holder was used as described before (62). In both cases linear heating was used from 10 or 20 to 90 °C. The ramp rate was 0.2 °C/min in the range of ± 20 °C of the Tm of the samples and 0.5 °C/min at other temperatures. Absorption values were measured at 0.5 °C intervals. Tm values were obtained from the melting curves with the use of the MeltWin program (63).

Enthalpy (-Delta HO) and entropy (-Delta SO) data for duplex formation of the 15-mers were obtained in two ways: from the 1/Tm - ln Ct plots (Fig. 3), where Ct is the total strand or duplex concentration, and from the melting profiles by using MeltWin, version 3.0. This program fits the shape of each curve to the two-state model with sloping base lines using a nonlinear least-square program (63). For the former method six duplex concentrations were used which ranged from 1 to 125 µM and measurements were carried out as described before (62). From the parameters obtained from the 1/Tm - ln Ct plots, only the -Delta HO values are shown in Table I. These were the averages of three to six determinations of separate samples. For the shape analysis with the melt curve processing program MeltWin the melting profiles were truncated at 20 and 90 °C.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3.   The 1/Tm versus ln Ct plots, determined in the range 1 to 125 µM total strand concentration (Ct), were used to calculate the transition enthalpy and entropy values for the 15-mer duplexes in the model-independent way. Solid symbols in the figure refer to the control duplexes, the open symbols are for the epsilon A-containing ones. Solid and open symbols of the same shape refer to the same tandem neighbors flanking A or epsilon A in the duplex. According to the numbers on the right these are: 1, for the duplex with the central sequence AAepsilon AAA; 2, TTepsilon ATT; 3, AAAAA; 4, TTATT; 5, GGepsilon AGG; 6, CCepsilon ACC; 7, GGAGG; and 8, CCACC.


    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Determination of the Extent and Rate of Cleavage of epsilon A-containing 15-mers by Human and Mouse APNG-- Preliminary data on extent of cleavage of four 25-mer epsilon A-containing oligomers with the same central sequences exhibited a definitive sequence dependence of cleavage of epsilon A when using human APNG (64). The order of cleavage efficiency was -GGepsilon AGG- or -CCepsilon ACC- > -AAepsilon AAA- or -TTepsilon ATT-. The sequence -GGepsilon ATT- in the same 25-mer gave an intermediate level of cleavage. The length of these duplexes was considered to be too long for precise determination of the role of thermal stability in repair. Therefore, in this study the same flanking doublets were kept and both ends of the 15-mers truncated by 5 nucleotides in the new substrates, yielding the 15-mer sequences shown under "Experimental Procedures" (Fig. 1).

In order to show both the extent and rate of repair of the 15-mer duplexes with epsilon A flanked by differing neighbor bases, cleavage with human APNG was determined as a function of enzyme concentration for 20 min (Fig. 4A) and of time using 2.5 ng of human APNG (Fig. 4B). The 20-min time and 2.5 ng of APNG, which was within the linear portion of these cleavage curves, was used for all subsequent experiments involving human APNG. A representative gel is shown in Fig. 5 which presents the cleavage of epsilon A-containing oligomers after reaction with APNG or APNG + HAP1. Note that lanes 5-8 have a double-band on cleavage presumably resulting from the beta -elimination mechanism due to high pH and high temperature in the treatment of samples (65, 66). When HAP1 is added to the APNG reaction (lanes 9-12) there is only a single band seen which is the result of cleavage of the phosphodiester bond 5' to the AP site (65). The 15-mers were in two classes in terms of repair efficiency: G·C or C·G neighbor pairing and A·T or T·A neighbor pairing. The A-containing control 15-mers were not cleaved in the presence of human APNG (data not shown). The epsilon A-containing duplexes with G·C or T·A flanking bases were cleaved by wild-type crude HeLa extracts which contain full-length human APNG and the same preference for oligomers with G·C pairs was found (Fig. 6). Similar results of differential cleavage were also obtained with a purified cloned mouse APNG (Fig. 7). APNG, as expected, did not cleave the four single-stranded epsilon A-containing 15-mers (data not shown).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 4.   A, the effect of increasing human APNG concentration on cleavage efficiency after 20 min at 37 °C. B, the effect of increasing time at 37 °C on cleavage efficiency using 2.5 ng of human APNG. The central 5-base sequences are shown in the figure. The symbols are GGepsilon AGG (black-square), CCepsilon ACC (black-down-triangle ), AAepsilon AAA (open circle ), and TTepsilon ATT (Delta ). The conditions for enzymatic reaction and quantitation are given under "Experimental Procedures."


View larger version (35K):
[in this window]
[in a new window]
  		Fig. 5.   Autoradiogram of cleavage of the four epsilon A-containing oligomers using buffer only (lanes 1-4), 2.5 ng of human APNG (lanes 5-8), and 2.5 ng of human APNG plus 25 ng of HAP1 (lanes 9-12). The positions of the cleavage products are indicated on the right. Note that both intact and cleavage product show different mobilities as a result of different neighbor tandem bases. All the reactions were performed at 37 °C for 20 min.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6.   Cleavage efficiency of epsilon A-containing oligomers using crude HeLa extract which contains wild-type human APNG. Reaction time is 20 min at 37 °C and the other reaction conditions are described under "Experimental Procedures." Only two representative oligomers are shown.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 7.   Cleavage efficiency of epsilon A-containing oligomers using mouse APNG as a function of protein concentration (0.5-5 ng, 20 min, 37 °C). The reaction conditions are the same as described in the legend to Fig. 4.

With either human or mouse APNG proteins, there was approximately a 3-5-fold preference for cleavage of oligomers with neighboring G·C or C·G pairs over A·T or T·A pairs (Figs. 4, 6, and 7). In order to observe whether complete cleavage of epsilon A-containing oligomers in all sequences could occur, both the time of reaction was increased from 20 to 60 min and the enzyme concentration increased up to 100 ng. Under these conditions, cleavage mediated by the human APNG reached saturation but not completion for all epsilon A-containing oligomers (Fig. 8). The remaining uncleaved oligomers still showed detectable differences in cleavage between oligomers with G·C or C·G and A·T or T·A flanking bases in the range of 40 to 80 ng of human APNG (Fig. 8).


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 8.   Persistence of intact epsilon A-containing 15-mer oligonucleotides after increasing amount of human APNG up to 100 ng and reaction time to 60 min. Note that in Fig. 4A the amount of APNG is up to 5 ng and the reaction time is 20 min. Under the conditions used in this figure the cleavage of oligomers containing GGepsilon AGG and CCepsilon ACC is almost maximized at 5 ng of enzyme and is about 3-fold more than that of oligomers containing AAepsilon AAA and TTepsilon ATT. From 40 to 80 ng of the enzyme concentration range, the difference between the two groups remains almost constant.

Melting Curves of 15-mer Duplexes and Thermodynamic Parameters-- All four epsilon A-containing duplexes and the analogous controls with A replacing epsilon A ("Experimental Procedures") were used to determine the thermodynamic stability of the annealed duplexes. Fig. 9 shows two representative melting curves. The percent denaturation of all four epsilon A-containing oligomers at 37 °C, derived from melting curves, is also shown in Fig. 9. It can be seen that the extent of denaturation at 37 °C of the oligomers with the central sequences -AAepsilon AAA- or -TTepsilon ATT- is about 3-fold more than that of -GGepsilon AGG- or -CCepsilon ACC-. The actual average of the Tm values of all oligomers from 3-6 runs is given in the first column of Table I. All these data indicate that the sequences differ significantly in the extent of double-strandedness at the temperatures used for repair studies.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 9.   Superimposed curves of two representative epsilon A-containing 15-mer oligomers (see Fig. 1 for complete sequences). The extent of denaturation at 37 °C (shown by the vertical line) is given in the figure for these two epsilon A-containing 15-mer oligomers. Calculated percent denaturation at 37 °C for all four epsilon A-containing oligomers is given in the figure. See Table I for additional Tm data and also the buffer conditions used.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Thermodynamic parameters for the melting of the 15-mer duplexes
The measurements were carried out in 0.1 M NaCl, 0.01 M sodium phosphate, and 0.1 mM EDTA (pH 7.0). The values shown are the averages of three to six separate determinations. The Tm values shown were determined at a duplex concentration of 8 µM. Standard deviation of the Tm values averaged ± 0.5 °C. The average standard deviation for thermodynamic parameters of the MeltWin analysis was ± 3.6 kcal/mol for Delta H°, ± 10.6 cal/mol · K for Delta S°, ± 0.21 kcal/mol for Delta G°37, and ± 3.1 kcal/mol for Delta H° obtained from the 1/Tm - ln Ct plots.

Table I also contains the transition enthalpy (Delta HO), entropy (Delta SO), and free energy (Delta GO37) values obtained by the MeltWin analysis of the melting profiles, as well as the enthalpy values obtained from the 1/Tm - ln Ct plots (Fig. 3). These plots show that the Tm values are dependent on the duplex concentration in a linear fashion indicating that molecularity of the transition was >1, except for the duplex with the AAAAA central sequence. With this latter duplex the concentration dependence was observed only from 1 to 30 µM, indicating a non-bimolecular melting transition above 30 µM. Enthalpy is calculated from the straight line obtained from 1 to 30 µM.

The enthalpy and entropy values obtained from the MeltWin curve fit and analysis are consequently lower in all cases than those obtained from the 1/Tm - ln Ct plots. In the case of duplexes with an epsilon A adduct the average difference is -22% (-17 to -28%). This indicates that the melting of the epsilon A-containing 15-mer duplexes studied is not a clear two-state transition (duplex to single stranded coils) (37, 67). Other less base paired intermediates, such as a "bubble," may be present in significant amount during the transition (68).

The Delta GO37 values show a similar trend to those shown by the Tm values, except for the duplex with GGAGG central sequence. The lower Delta GO37 value obtained can be the result of the GGGGAGGG sequence in this oligomer. Three or more consecutive G bases have been found to yield lower enthalpy values than expected (37). In the sequence used there are both four and three consecutive G bases present on each side of A which could lead to similar results.

The Delta GO37 values for the epsilon A-containing duplexes indicate that the duplexes with GGepsilon AGG and CCepsilon ACC central sequences are more stable, by an average of 3.3 kcal/mol, than the duplexes with AAepsilon AAA and TTepsilon ATT central sequences. The -Delta Delta GO values, however, show little dependence on sequence, similar to the -Delta Tm values. The epsilon A substitution decreases both the transition enthalpy and entropy values similarly in all four sequences (Table I).

Effect of Temperature Range and Shift on Cleavage of epsilon A-containing Oligomers-- Based on data in Fig. 9 and Table I it was clear that, at 37 °C, oligomers with epsilon A flanked by A·T or T·A pairs were less stable than oligomers with epsilon A flanked by G·C or C·G pairs, which have a higher Tm. These data would seem to correlate with the differences in repair at 37 °C (Figs. 4, 6, and 7). Thus, an experiment using APNG in which the temperature range 0-45 °C was used to confirm this presumed relationship between thermodynamic properties and repair efficiency, since a double-stranded DNA substrate is needed for repair by this enzyme.

The data obtained are shown in Fig. 10 which substantiates the above premise. At temperatures 30 °C and below, the cleavage of the epsilon A-containing oligomers with the two types of pairs is not significantly different. However, as the temperature of the reaction increases, the oligomers containing G·C or C·G pairs were cleaved increasingly and the percent cleavage reached 50-70% at 45 °C while the A·T or T·A containing oligomers actually showed decreased repair at 40 and 45 °C (below 10%) compared with 30 °C.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 10.   Effect of reaction temperature on cleavage efficiency of epsilon A-containing oligomers with different tandem neighboring groups. Each point represents an independent reaction mixture incubated for 20 min with 2.5 ng of human APNG protein at the indicated temperature. The normal 37 °C physiological temperature is shown by a dashed vertical line. At this temperature and above there is a dramatic difference in cleavage between the oligomers with GGepsilon AGG and CCepsilon ACC and oligomers containing AAepsilon AAA or TTepsilon ATT.

In Fig. 11, the question was addressed of whether the poor enzyme activity toward all four oligomers at 10-20 °C (Fig. 10) was due to low enzymatic activity at low temperatures, which might obscure any real differences in cleavage between the A·T and G·C pairs. In Fig. 11, we illustrate a time and temperature shift experiment in which either 20 or 100 min incubation of all four epsilon A-containing oligomers at 20 °C was followed by an additional 20 min at 37 °C. The primary observation is that 20 °C incubation for 100 min is, as expected, more effective than the 20-min incubation as reflected by percent cleavage. However, the differences in cleavage between sequences are minimal, which reflects the similar thermal stability of these oligomers at low temperature, as shown in Fig. 9. In addition, regardless of whether 20 °C incubation is for 20 or 100 min (Fig. 11), the shift to 37° from 20 °C increased the rate of cleavage of oligomers with G·C or C·G more than that of oligomers containing A·T or T·A. This illustrates that the lower thermal stability conferred by A·T or T·A base pairing at higher temperature can be responsible for the poor excision of epsilon A in an A·T environment compared with that found in a G·C environment.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 11.   Effect of temperature shift on cleavage efficiency of epsilon A-containing oligomers with four different neighbor groups. Reactions were initiated at 20 °C for the indicated times (20 or 100 min) with 2.5 ng of human APNG. Following this, half of each sample was placed in a 37 °C water bath and incubated for a further 20 min. The symbols for the four oligomers are shown in the figure with only one tandem pair. Note that regardless of whether the 20 °C incubation was for 20 or 100 min, differences in cleavage were minimal, while the 100-min incubation showed the expected time dependence.


    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

There are numerous interactions in biochemistry in which the sequence context of nucleic acids or proteins influences the results. Whenever sequence context is implicated there is also a structural factor which is not always apparent.

There is a considerable body of data indicating that modification, replication, and repair of nucleic acids are not random. The role of strandedness and stacking interactions of bases have been studied in many contexts and are implicated in selection of favored sites or reaction rates, but differ in specificity for each type of reaction. Thus, experiments which are designed to probe, in this case, repair specificity as a function of sequence context, need to be simplified and a single question addressed. This type of study is facilitated by using DNA glycosylases which are monomeric and have no absolute requirement for cofactors.

In our previous studies on in vitro replication in a defined system, we found a strong effect of the immediate sequence 3' to the adducts studied (14, 19). Goodman and colleagues (17) reported a 5' sequence effect in replication. The polymerase caused a mismatch at a higher frequency when the 5' base on the primer to the incoming base was a purine. This is attributed to better stacking interactions (17). In another type of experiment, Singer and colleagues (14, 69) found that the base following a m6G mismatch in the template strand is also important for extension kinetics.

When repair is considered, with noted exceptions, a double-stranded DNA is the preferred substrate (41). In our system we systematically examined simultaneously the effect of both 3' and 5' bases adjacent to epsilon A. In order to increase both base stacking and base pairing we used tandem bases 3' and 5' to this adduct, which does not form a Watson-Crick base pair with the opposite T as reported for the NMR structure of an 11-mer containing epsilon A·T (70).

In the present study we explored a relationship between the thermodynamic stability of the oligomer duplexes containing epsilon A and the sequence-dependent glycosylase repair of this adduct. The thermodynamically more stable duplexes with G·C or C·G doublet base pairs flanking the epsilon A are repaired with higher rate above 30 °C than are the less stable duplexes where A·T or T·A doublet pairs flank the epsilon A. In Fig. 10 we show that the adduct is repaired with a constantly increasing rate from 30 to 45 °C when the flanking sequences are G·C/C·G doublet pairs, while repair of epsilon A decreases above 30 °C with A·T/T·A flanking pairs. The experimental melting curves in Fig. 9 also show that the difference in the extent of denaturation between G·C and A·T flanking pairs containing duplex actually begins to increase considerably above 30 °C. Therefore, the similarity between the extent of denaturation shown by the melting curves (Fig. 9) and temperature dependence of repair (Fig. 10) suggests a qualitative correlation. This relationship is valid if the experimental melting curve represents a physical property which may be important in the repair and the conditions are similar in both types of experiments.

It should be assumed that there is a partially "open" structure in the vicinity of the adduct. Evidence that supports the open structure comes from the thermodynamic data. A comparison of enthalpies obtained by two different methods (Table I) shows that the melting transition of the epsilon A-containing duplexes is not a clear two-state process. Very similar findings were described for single base mismatches (68). In this situation, melting intermediates can be present in a partially denatured form, such as a bubble or loop structure (68) in the vicinity of an epsilon A·T region. Therefore, melting curves indicate not only the ratio of duplex to single strands at any temperature but also show the contribution of bubbles of various length to the increase of absorption in the absorption versus temperature curves.

The extent of denaturation, that is, thermal stability, should be similar under the conditions used for repair and denaturation. Although duplex concentrations are lower in the enzyme reaction than in the thermal transition measurements, the Tm decrease originating from the lower duplex concentration is essentially compensated by the higher ionic strength used in the enzyme assay (data not shown). Thus melting curves can be used for a qualitative comparison with repair data.

Quantitation is, however, difficult. Melting curves observe the whole duplex molecule not only in the vicinity of the epsilon A-containing part which may be important for the enzyme and interesting for the proposed correlation. Extent of denaturation depends on the chain length of a duplex. For example, 25-nucleotide long duplexes with the same central 15-mer sequence used in this work have higher Tm values and lower denaturations at 37 °C than the 15-mers (Fig. 9).2 However, the ratio for the extent of denaturation for duplexes with GGepsilon AGG and TTepsilon ATT sequences is similar with both chain lengths. Therefore, in the vicinity of epsilon A, where the bubbles or loops may be present, the actual extent of denaturation should be much higher than that measured for the full-length 15-mer duplexes. As denaturation occurs in the immediate vicinity of epsilon A, probably as a bubble, repair is decreased. Since extent of bubble formation is a function of thermodynamic stability, repair efficiency, and thermodynamic stability are interrelated.

These data would indicate that at physiological temperature (37 °C) such adducts as epsilon A, which require a double strand for their repair, would be less repairable, or more persistent in an A·T flanking region than in a G·C flanking region, since glycosylase-catalyzed removal of epsilon A is the initial rate-limiting step in repair. There are many factors in vivo that can affect the mutation rate. From this work and that of others, the inefficient DNA repair in specific sequence contexts could be a contributing factor for the accumulation of genetically harmful DNA lesions, implicated in carcinogenesis and aging.

    ACKNOWLEDGEMENTS

We are indebted to Dr. T. R. O'Connor for the human APNG, to Drs. R. Roy and S. Mitra for the mouse APNG, and Dr. I. D. Hickson for HAP1. We also thank Dr. D. H. Turner for providing the MeltWin program (version 3.0) for analysis of melting profiles and Dr. M. Burkard for helpful instructions. We thank Michael Medina for expert technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA 47723 and CA 72079 (to B. S.) and was administered by the Lawrence Berkeley National Laboratory under Department of Energy contract DE-AC03-76SF00098.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 510-642-0637; Fax: 510-486-6488.

The abbreviations used are: APNG, alkylpurine-DNA-N-glycosylase; HAP1, human AP endonuclease 1; epsilon A, 1,N6-ethenoadenine; AP site, abasic site.

2 B. Hang, J. Sági, and B. Singer, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Haseltine, W. A., Gordon, L. K., Lindan, C. P., Grafstrom, R. H., Shaper, N. L., and Grossman, L. (1980) Nature 285, 634-641[CrossRef][Medline] [Order article via Infotrieve]
  2. Burns, P. A., Gordon, A. J. E., and Glickman, B. W. (1987) J. Mol. Biol. 194, 385-390[CrossRef][Medline] [Order article via Infotrieve]
  3. Dolan, M. E., Oplinger, M., and Pegg, A. E. (1988) Carcinogenesis 9, 2139-2143[Abstract/Free Full Text]
  4. Richardson, F. C., Boucheron, J. A., Skopek, T. R., and Swenberg, J. A. (1989) J. Biol. Chem. 264, 838-841[Abstract/Free Full Text]
  5. Carothers, A., Urlaub, G., Mucha, J., Harvey, R., Chasin, L. A., and Grunberger, D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5464-5468[Abstract/Free Full Text]
  6. Skopek, T. R., Walker, V. E., Cochrane, J. E., Craft, T. R., and Cariello, N. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7866-7870[Abstract/Free Full Text]
  7. Cmarik, J. L., Humphreys, W. G., Bruner, K. L., Lloyd, R. S., Tibbetts, C., and Guengerich, F. P. (1992) J. Biol. Chem. 267, 6672-6679[Abstract/Free Full Text]
  8. Ross, H., Bigger, C. A. H., Yagi, H., Jerina, D. M., and Dipple, A. (1993) Cancer Res. 53, 1273-1277[Abstract/Free Full Text]
  9. Yarema, K. J., Wilson, J. M., Lippard, S. J., and Essigmann, J. M. (1994) J. Mol. Biol. 236, 1034-1048[CrossRef][Medline] [Order article via Infotrieve]
  10. Wyatt, M. D., Lee, M., Garbiras, B. J., Souhami, R. L., and Hartley, J. A. (1995) Biochemistry 34, 13034-13041[CrossRef][Medline] [Order article via Infotrieve]
  11. Petruska, J., and Goodman, M. F. (1985) J. Biol. Chem. 260, 7533-7539[Abstract/Free Full Text]
  12. Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415-14423[Abstract/Free Full Text]
  13. Singer, B., Chavez, F., Goodman, M. F., Essigmann, J. M., and Dosanjh, M. K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8271-8274[Abstract/Free Full Text]
  14. Dosanjh, M. K., Galeros, G., Goodman, M. F., and Singer, B. (1991) Biochemistry 30, 11595-11599[CrossRef][Medline] [Order article via Infotrieve]
  15. Cai, H., Bloom, L. B., Eritja, R., and Goodman, M. F. (1993) J. Biol. Chem. 268, 23567-23572[Abstract/Free Full Text]
  16. Bloom, L. B., Otto, M. R., Beechem, J. M., and Goodman, M. F. (1993) Biochemistry 32, 11247-11258[CrossRef][Medline] [Order article via Infotrieve]
  17. Bloom, L. B., Otto, M. R., Eritja, R., Reha-Krantz, L. J., Goodman, M. F., and Beechem, J. M. (1994) Biochemistry 33, 7576-7586[CrossRef][Medline] [Order article via Infotrieve]
  18. Goodman, M. F., Cai, H., Bloom, L. B., and Eritja, R. (1994) Ann. N. Y. Acad. Sci. 726, 132-142[Medline] [Order article via Infotrieve]
  19. Rao, S., Chenna, A., Slupska, M., and Singer, B. (1996) Mutat. Res. 356, 179-185[Medline] [Order article via Infotrieve]
  20. Hashim, M. F., and Marnett, L. J. (1996) J. Biol. Chem. 271, 9160-9165[Abstract/Free Full Text]
  21. Litinski, V., Chenna, A., Sági, J., and Singer, B. (1997) Carcinogenesis 18, 1609-1615[Abstract/Free Full Text]
  22. Jones, M., Wagner, R., and Radman, M. (1987) Genetics 115, 605-610[Abstract/Free Full Text]
  23. Seeberg, E., and Fuchs, R. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 191-194[Abstract/Free Full Text]
  24. Foley, C. K., Pedersen, L. G., Darden, T. A., Glickman, B. W., and Anderson, M. W. (1991) Mutat. Res. 255, 89-93[Medline] [Order article via Infotrieve]
  25. Mu, D., Bertrand-Burggraf, E., Huang, J.-C., Fuchs, R. P. P., and Sancar, A. (1994) Nucleic Acids Res. 22, 4869-4871[Abstract/Free Full Text]
  26. Eftedal, I., Volden, G., and Krokan, H. E. (1994) Ann. N. Y. Acad. Sci. 726, 312-314[Medline] [Order article via Infotrieve]
  27. Bender, K., Federwisch, M., Loggen, U., Nehls, P., and Rajewsky, M. F. (1996) Nucleic Acids Res. 24, 2087-2094[Abstract/Free Full Text]
  28. Sander, M., and Benhaim, D. (1996) Nucleic Acids Res. 24, 3926-3933[Abstract/Free Full Text]
  29. Sibghat-Ullah, Gallinari, P., Xu, Y.-Z., Goodman, M. F., Bloom, L. B., Jiricny, J., and Day, R. S., III (1996) Biochemistry 35, 12926-12932[CrossRef][Medline] [Order article via Infotrieve]
  30. Delagoutte, E., Bertrand-Burggraf, E., Dunand, J., and Fuchs, R. P. P. (1997) J. Mol. Biol. 266, 703-710[CrossRef][Medline] [Order article via Infotrieve]
  31. Mekhovich, O., Tang, M-s., and Romano, L. J. (1998) Biochemistry 37, 571-579[CrossRef][Medline] [Order article via Infotrieve]
  32. Hang, B., Chenna, A., Sági, J., and Singer, B. (1998) Carcinogenesis 19, 1339-1343[Abstract/Free Full Text]
  33. Nelson, J. W., Martin, F. H., and Tinoco, I., Jr. (1981) Biopolymers 20, 2509-2531[CrossRef][Medline] [Order article via Infotrieve]
  34. Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3746-3750[Abstract/Free Full Text]
  35. Petruska, J., Goodman, M. F., Boosalis, M. S., Sowers, L. C., Cheong, C., and Tinoco, I., Jr. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6252-6256[Abstract/Free Full Text]
  36. Ke, S.-H., and Wartell, R. M. (1995) Biochemistry 34, 4593-4600[CrossRef][Medline] [Order article via Infotrieve]
  37. SantaLucia, J., Jr., Allawi, H. T., and Seneviratne, P. A. (1996) Biochemistry 35, 3555-3562[CrossRef][Medline] [Order article via Infotrieve]
  38. Allawi, H. T., and SantaLucia, J., Jr. (1998) Biochemistry 37, 2170-2179[CrossRef][Medline] [Order article via Infotrieve]
  39. Shibutani, S., Margulis, L. A., Geacintov, N. E., and Grollman, A. P. (1993) Biochemistry 32, 7531-7541[CrossRef][Medline] [Order article via Infotrieve]
  40. Arghavani, M. B., SantaLucia, J., and Romano, L. J. (1998) Biochemistry 37, 8575-8583[CrossRef][Medline] [Order article via Infotrieve]
  41. Lindahl, T. (1982) Annu. Rev. Biochem. 51, 61-87[CrossRef][Medline] [Order article via Infotrieve]
  42. Demple, B., Jacobsson, A., Olsson, M., Robins, P., and Lindahl, T. (1982) J. Biol. Chem. 257, 13776-13780[Abstract/Free Full Text]
  43. Scicchitano, D., and Pegg, A. E. (1982) Biochem. Biophys. Res. Commun. 109, 995-1001[CrossRef][Medline] [Order article via Infotrieve]
  44. Singer, B., Antoccia, A., Basu, A. K., Dosanjh, M. K., Fraenkel-Conrat, H., Gallagher, P. E., Kusmierek, J. T., Qiu, Z.-H., and Rydberg, B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9386-9390[Abstract/Free</