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© 1995 Oxford University Press 4111-4117

Footnote

Differential effects of the incorporation of 1-(2-deoxy-2-fluoro- [beta]-D-arabinofuranosyl)-5-iodouracil (FIAU) on the binding of the transcription factors, AP-1 and TFIID, to their cognate target DNA sequences

Differential effects of the incorporation of 1-(2-deoxy-2-fluoro- [beta]-D-arabinofuranosyl)-5-iodouracil (FIAU) on the binding of the transcription factors, AP-1 and TFIID, to their cognate target DNA sequences K. A. Staschke , K. K. Richardson , T. E. Mabry , A. J. Baxter , J. C. Scheuring , D. M. Huffman , W. C. Smith , F. C. Richardson and J. M. Colacino*

Lilly Research Laboratories, Indianapolis , IN 46285-0438, USA

Received August 22, 1996 ; Revised and Accepted September 19, 1996

ABSTRACT

The thymidine analog, 1-(2-deoxy-2-fluoro- [beta] -D-arabinofuranosyl)-5-iodouracil (FIAU), is incorporated into DNA in cell culture and in vivo . To investigate the effect of incorporation of FIAU into DNA on the binding of transcription factors, oligonucleotide duplexes which bind specifically to activator protein 1 (AP-1) or to TFIID were synthesized and binding of these oligonucleotides to their respective proteins was studied using gel-shift analysis. When thymidine at position -3, -1, 1 or 7 (relative to the first thymidine of the core binding sequence) was replaced with FIAU, binding to AP-1 was ~ 82, 28, 86 and 51%, respectively, of the binding to the non-substituted oligonucleotide to AP-1. When thymidine at position 3 or 5 (each adjacent to the center of dyad symmetry) was replaced with FIAU, binding to AP-1 was abrogated. Oligonucleotides containing FIAU at positions -1, 3 or 5, were much less able to compete with radiolabeled wild-type oligonucleotides for binding to AP-1. In contrast, the presence of FIAU, depending on its location, resulted in the increased binding of TFIID to its consensus target DNA sequence. These results indicate that incorporation of FIAU into DNA may induce local conformational changes resulting in the altered ability of transcriptional factors to bind to their cognate DNA sequences. Additional studies demonstrated that the presence of FIAU at a position 5 ' to the cleavage site in the consensus sequence T*TAA (where * is the cleavage site) inhibited restriction of the oligomeric duplex by Mse I.

INTRODUCTION

The activation or repression of gene expression is dependent upon the recognition of specific DNA sequences by their cognate transcriptional regulatory proteins. Recognition of target DNA sequences involves the ability of proteins to `read' local DNA topography ( 1 ). Three well studied examples of protein-DNA interactions include: the binding of the activator protein 1 (AP-1) to the palindromic TPA response element (TRE) ( 2 , 3 ); the binding of the trp repressor protein to its operator sequence ( 4 ); and the binding of TFIID to the sequence TATAAAA (as reviewed in 5 ).

The thymidine analog FIAU [1-(2-deoxy-2-fluoro-[beta]-D-arabinofuranosyl)-5-iodouracil is a potent inhibitor of hepatitis B virus (HBV) replication in cell culture ( 6 , 7 ) and in the woodchuck model of HBV infection ( 8 ). FIAU contains an intact 3'-OH and is able to incorporate into nuclear DNA in vitro ( 9 - 13 ) and in vivo ( 14 ). Additionally, FIAU is able to incorporate into mitochondrial DNA ( 12 , 13 ) and duck hepatitis B virus primer DNA ( 15 ). It has been shown that duplex DNA containing monofluoronucleotides has an altered conformation ( 16 ). Therefore, incorporation of a halogenated nucleoside analog, such as FIAU, into DNA might be expected to alter DNA topography leading to the disruption of the protein-DNA interactions. To investigate the potential for FIAU to affect interactions between proteins and DNA, we synthesized oligonucleotides containing the consensus sequence for binding to AP-1 or TFIID using FIAU as a substitute for thymidine in various nucleotide positions. The ability of these transcription factors to bind to oligonucleotides containing FIAU was examined by gel-shift analysis.

Previously published work ( 17 ) demonstrated that the incorporation of the nucleoside analog, 2',2'-difluorodeoxycytidine (dFdC) into the recognition sequence for Bam HI did not alter, appreciably, the sensitivity of the oligonucleotide duplex to cleavage by this restriction endonuclease. However, incorporation of dFdC into the recognition sequence at a position 5' to the cleavage site of Kpn I did decrease the sensitivity of the oligonucleotide duplex to this restriction endonuclease. Therefore, experiments were conducted to determine the effects of the presence of FIAU on the susceptibility of substituted oligonucleotides to cleavage by the restriction endonuclease Bam HI or to cleavage by Mse I, which cleaves DNA between thymidine residues.

MATERIALS AND METHODS

Preparation of nuclear extracts


Figure 1 . The effect of FIAU on the binding of the transcription factor AP-1 to its target DNA sequence. ( A ) The nucleotide sequence of the DNA probe containing the consensus AP-1 binding site which was used in this study. The AP-1 binding site is boxed and the center of dyad symmetry is indicated by a dot. The thymidine nucleotides which were substituted with FIAU are indicated in bold. ( B) Binding of AP-1 was assayed using a non-substituted consensus AP-1 or FIAU-substituted probe, analyzed by gel electrophoresis, and exposed to X-ray film as described in Materials and Methods. The results of a representative experiment are shown. ( C ) The autoradiogram in (B) was scanned using a Bio Image Whole Band Analyzer and the extent of AP-1 binding to the consensus AP-1 probe was set to 100%. The extent of AP-1 binding to each probe was normalized to the specific activity of that particular probe. Each value represents the average of three independent experiments.

Nuclear extracts from HeLa cells were prepared according to the protocol of Prywes and Roeder ( 18 ) with modifications. Briefly, subconfluent HeLa cells (five 225 cm 2 tissue culture flasks) were washed with cold calcium and magnesium free phosphate buffered saline (CMF-PBS) and then harvested by scraping. The cells were pelleted by centrifugation at 500 g for 5 min. The pellet was washed with 5 ml of cold CMF-PBS followed by 5 ml of ice cold hypotonic swelling buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF). The pellet was then resuspended in 5 ml of cold hypotonic swelling buffer and the cells were allowed to swell on ice for 10 min. The cells were homogenized by 40-60 strokes of a Dounce homogenizer and the nuclei were pelleted by centrifugation at 1000 g for 5 min. The nuclei were resuspended in 300 [mu]l of cold nuclear resuspension buffer (20 mM HEPES pH 7.9, 20% glycerol, 1.5 mM MgCl 2 , 0.5 mM DTT, 0.2 mM PMSF) and 4 M KCl was added to a final concentration of 0.3 M KCl. The nuclei were rocked at 4oC for 30 min, then centrifuged at 13 000 g for 15 min. The supernatant was dialyzed against 500 ml of BC100 (20 mM HEPES pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF) for 2 h at 4oC. The dialysate was centrifuged at 13 000 g for 15 min at 4oC and the supernatant was divided into aliquots and stored at -70oC until used. The protein content of the nuclear extract was determined using a protein assay kit (BioRad, Richmond, CA).

Oligonucleotides and preparation of radiolabeled probes: AP-1

Unsubstituted oligonucleotides were synthesized on a Beckman Oligo 1000 DNA synthesizer (Beckman, Fullerton, CA). Oligonucleotides containing FIAU were synthesized on an ABI 381A DNA synthesizer (ABI, Foster City, CA). The FIAU phosphoramidite was synthesized according to Richardson et al . ( 17 ). For FIAU-containing oligonucleotides, the coupling time was lengthened from 15 to 300 s to ensure efficient coupling. Oligonucleotides were purified by OPCtm column chromatography as specified by the manufacturer (Clontech, Palo Alto, CA). The nucleotide sequence of the AP-1 oligonucleotide used in this study as well as the positions of the FIAU substitutions are shown in Figure 1 A. To form double-stranded oligonucleotides each oligonucleotide was annealed to its complementary oligonucleotide in a buffer containing 20 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , 50 mM NaCl. The formation of duplex DNA was verified by gel electrophoresis (data not shown). Double-stranded oligonucleotides were end-labelled with 32 P in a reaction mixture consisting of 3.5 pmol of double-stranded oligonucleotide, 70 mM Tris-HCl pH 7.6, 10 mM MgCl 2 , 50 mM DTT, 10 mCi [[gamma]- 32 P]ATP (sp. act. 3000 Ci/mmol) and 10 U of T4 polynucleotide kinase in a total volume of 10 [mu]l. Unincorporated radiolabel was removed using Sephadex G-25 spin columns. The radiolabelled probes ranged in specific activity from 76 000 to 110 000 c.p.m./ng.

Electrophoretic mobility shift assays: AP-1

The binding reaction mixture consisted of 10-15 [mu]g of HeLa cell nuclear extract, 10 mM Tris-HCl pH 7.5, 4% glycerol, 1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 0.05 mg/ml poly(dI-dC) . poly(dI-dC) and 32 P-labelled probe (~0.5 ng) in a final volume of 10 [mu]l. Reactions were incubated at room temperature for 30 min and then loaded onto a 4% nondenaturing polyacrylamide gel and electrophoresed in 1* Tris-glycine-EDTA buffer (50 mM Tris, 0.38 mM glycine, 2.1 mM EDTA). Following electrophoresis, gels were dried under vacuum. Dried gels were exposed to X-ray film and quantified using a Bio Image Whole Band Analyzer (Millipore, Bedford, MA). For competition studies, binding reactions were incubated with cold competitor oligonucleotides for 10 min prior to addition of the 32 P-labelled probe. The amount of cold competitor required to reduce binding by 90% was determined by the method of Reed and Muench ( 19 ).

Electrophoretic mobility shift assays: TFIID

Gel retardation studies were conducted, in general, following methods described ( 20 ). Ten pmol of duplexed oligomer were end-labeled with 32 P using T4 polynucleotide kinase (New England Biolabs) following established procedures and unincorporated radiolabel was removed using Sephadex G-25 spin columns. Binding of TFIID oligonucleotide duplexes was carried out by incubating 2 pmol of duplex oligomer in a total of 5 [mu]l containing 1 footprint unit (f.p.u.) of TFIID (Promega Corp., Madison, WI), 2.5 [mu]l binding buffer (40 mM HEPES pH 7.9, 50 mM KCl, 4 mM spermidine, 0.2 mM EDTA, 0.05% NP-40, 20% glycerol, 1.0 mM dithiothreitol, 200 [mu]g/ml BSA) and 1.5 [mu]l water. Binding was allowed to occur over 40 min at room temperature after which 2 [mu]l of glycerol were added and the entire sample was used for electrophoresis. Oligonucleotides bound to TFIID were separated from unbound oligonucleotides on 4% polyacrylamide (40:1 acrylamide:bis-acrylamide, running buffer: 0.025 M Tris-HCl pH 8, 0.19 M glycine, 1 mM EDTA and 50 mM MgCl 2 ) ( 21 ). Gels were electrophoresed for 90 min at 30 mA after which time they were transferred to Whatman DEAE paper and dried under vacuum.

The extent of binding, as indicated by complex formation, was quantified using a Molecular Dynamics Phosphorimager (Sunnydale, CA). The radioactivity in each spot was indexed using the volume measurement. To account for loading differences the extent of gel retardation was calculated as follows:

(volume bound oligomer ) - (volume background ) / (volume unbound oligomer )

The TFIID oligomeric duplexes used in these studies are shown in Figure 3 A.

The effects of FIAU modifications on TFIID binding were evaluated using a two-factor analysis of variance (ANOVA) with each oligomer as an experimental treatment effect and each replicate as a blocking factor. A square root transformation of the measured response values was made prior to analysis in order to satisfy the ANOVA assumptions of normality and variance homogeneity. Mean responses for FIAU-modified oligos were compared pairwise with the mean response for the unmodified oligo using Dunnett's procedure ( 22 ). All tests were completed using the MIXED Procedure ( 23 ) and a nominal 0.05 significance level. Restriction enzyme digestion

Duplex oligomer (0.33 pmol) containing the restriction enzyme sequence for Mse I (Table 3 ) and 5'-end-labeled with 32 P were digested with 1 [mu]l Mse I (4000 U/ml), 1 [mu]l reaction buffer (New England Biolabs, Beverly, MA) and water q.s. to 10 [mu]l. Similarly, duplex oligomers (0.33 pmol) containing the restriction enzyme sequence for Bam HI (Table 3 ) and 5'-end-labeled with 32 P were digested with 1 [mu]l Bam HI (10 000 U/ml) and 1 [mu]l reaction buffer B (Boehringer Mannheim, Indianapolis, IN). Water was added to all samples q.s. to 10 [mu]l. Digestions were allowed to proceed for 180 min after which time little or no further digestion was observed (data not shown). The reactions were stopped by addition of 5 [mu]l Stop Solution (USB, Cleveland, OH) followed by quick freeze in a dry ice-isopropanol bath.

Five microliters of each labeled digested duplex oligomer were electrophoresed on a 20% polyacrylamide gel along with 5 [mu]l of labeled undigested duplex oligomer. The gels were then scanned into a Molecular Dynamics Phosphorimager and the amount of radioactivity associated with the uncut oligomer and each restriction fragment was quantitated in phosphorimaging units. The units associated with the uncut oligomer were divided by total units associated with both uncut and restriction oligomers. The quotient was multiplied by 100 to obtain the percentage of uncut oligomer remaining after 180 min. Percentage changes were used to determine the extent of enzyme digestion.

RESULTS AND DISCUSSION

Gel shift analysis of AP-1 binding to non-substituted and FIAU-substituted duplex DNA

The toxicity of a particular nucleoside analog is a function of its metabolism and the extent to which it is incorporated into nuclear DNA, mtDNA, or both (reviewed in 24 ). The cytotoxicities of 9-[beta]-D-arabinofuranosyl-2-fluoroadenine ( 25 ) or 1-[beta]-D-arabinofuranosylcytosine ( 26 ) have been shown to be correlated directly with the incorporation of these nucleoside analogs into DNA. Previous studies ( 9 - 14 ) have demonstrated that FIAU is able to incorporate into cellular DNA. Furthermore, a linear relationship between the amount of FIAU incorporated into U-937 or MOLT-4 cellular DNA at 24 h and cytotoxicity at 72 h was established ( 11 ). To study the potential implications of such incorporation, we used a model system in which oligonucleotides representing the TPA response element (TRE) that binds specifically to activator protein 1 (AP-1) ( 2 , 3 ) were synthesized with FIAU in place of thymidines at various positions in the AP-1 binding sequence. The oligonucleotides corresponding to the top and bottom strands of the AP-1 consensus sequence containing FIAU at the indicated thymidine positions are shown in Figure 1 A. Each oligonucleotide was annealed to its complementary oligonucleotide and the resulting double-stranded DNA duplexes were end-labeled with 32 P and incubated with HeLa cell nuclear extracts as described in the Materials and Methods. Binding of AP-1 to the end-labeled DNA was evaluated by gel shift analysis. In Figure 1 B, binding of AP-1 to the non-substituted oligonucleotide duplex DNA is shown in the lane marked AP-1. When thymidine at position -3, -1, 1 or 7 was replaced with FIAU, binding to AP-1 was 82, 28, 86 and 51%, respectively, that of the binding of AP-1 to the non-substituted oligonucleotide duplex. When thymidine at position 3 or 5, each directly adjacent to the center of dyad symmetry of the TRE, was replaced by FIAU, binding to AP-1 was completely inhibited (Fig. 1 B and C). Thus, when FIAU replaces thymidine at positions within the sequence critical for binding to AP-1, binding is dramatically reduced. Although the most profound effects on AP-1 binding to the TRE were seen with FIAU substitutions at positions 3 and 5 of the TRE, when FIAU was present at position -1 of the TRE, binding to AP-1 was also inhibited. While it is not clear why this substitution has such an effect on AP-1 binding, it has been suggested that nucleotides outside the core binding sequence may influence the affinity of AP-1 for the TRE ( 27 ). Substitutions of FIAU at positions -3, 1 or 7 of the TRE resulted in little or no effect on binding to AP-1.

Binding of AP-1 to its target sequence in the presence of FIAU-containing cold competitor DNAs

Binding of AP-1 to its target DNA sequence was carried out in the presence of non-substituted or substituted cold competitor DNAs as described in Materials and Methods. As shown in Figure 2 A, with the exception of -3F, cold competitor DNA containing FIAU was less able to compete with end-labeled DNA for binding to AP-1. The extent of binding, as indicated by complex formation, was quantified by densitometric scanning (Fig. 2 B) and the relative affinity of each cold competitor DNA was calculated (Table 1 ). Cold competitor DNAs with FIAU in the 3 or 5 position displayed the least relative affinity for binding to AP-1, 0.2 and <0.1, respectively. Cold competitor DNAs with FIAU in positions -1, 1 or 7 displayed intermediate affinities and substitution of thymidine with FIAU at position -3 did not affect the affinity of the substituted oligonucleotide for AP-1.


Figure 2 . Binding of AP-1 to its target sequence in the presence of FIAU-containing cold competitor DNAs. ( A ) AP-1 binding was assayed in the presence of increasing amounts (0, 1-, 3-, 6-, 12- and 24-fold molar excess) of cold FIAU-substituted competitor DNAs as described in Materials and Methods. ( B ) The autoradiograms in (A) were scanned and the extent of AP-1 binding was calculated and plotted. Each point represents the average of duplicate samples.

Table 1 Results of competition studies with FIAU-substituted cold competitor DNAs
Cold competitor a

IC 90 b

Relative affinity c

AP-1

3.45

1.0

-3F

2.93

1.2

-1F

11.90

0.3

1F

5.48

0.6

3F

22.88

0.2

5F

>24.00

<0.1

7F

6.44

0.5

a For location of each FIAU substitution, see Figure 1A. b The amount of cold competitor (in fold molar excess) required to reduce AP-1 binding by 90%. The IC 90 values were determined by the method of Reed and Muench (1938). c Relative affinity was calculated by dividing the IC 90 of the AP-1 probe (3.45) by the IC 90 of each probe containing an FIAU substitution.

Effect of FIAU on TFIID binding


Figure 3 . The effect of FIAU on the binding of the transcription factor TFIID to its target DNA sequence. ( A ) The sequence of each oligonucleotide where F indicates the substitution of thymidine with FIAU. (B ) Phosphorimage of a gel (replicate 2, Table 2) in which binding of TFIID to target duplex oligonucleotide DNAs was evaluated as described in Materials and Methods. The slower migrating bands represent TFIID bound to the 32 P-end-labeled duplex oligonucleotide. The unbound 32 P-labeled oligonucleotides migrate to the bottom of the gel. The letter above each lane of the gel refers to the corresponding duplex as shown in (A).

In general, the presence of FIAU resulted in the increased binding of TFIID in a manner that was dependent on the location of the FIAU in the consensus sequence (Fig. 3 ). Compared with the control oligomer J, the FIAU-substituted oligomers L, S and V displayed 2.5-, 1.8- and 4-fold significantly greater binding to TFIID, respectively (Table 2 ). The greatest enhancements of binding to TFIID were observed when FIAU was located at the 5'-end of the consensus binding site. Although binding appeared less with oligomers T, Y (Table 2 ) and X (Fig. 3 ), these changes were not significant. The enhancing effect of FIAU on the binding of the oligonucleotide to TFIID is in contrast with the inhibitory effect the presence of FIAU had on the binding of AP-1. The reason for these apparently disparate results is not known, but may be due to differences between TFIID and AP-1 in the way each interacts with DNA. TFIID interacts with bases in the minor groove of DNA ( 28 ) whereas key amino acids of AP-1 may make contact with several nucleotides in addition to the 5-methyl groups of thymines located in the major groove as has been described for GCN4, a member of a family of eukaryotic transcription factors which includes AP-1 ( 29 ). The consequences of FIAU-mediated increased binding of TFIID are unknown but one result could be an increase in gene expression. Studies with yeast TFIID have shown that single substitutions in the TATAAA(A) consensus sequence resulted in greater transcriptional activity ( 30 ). Whether the placement of an FIAU in the TATAAA(A) sequence can have a similar effect remains to be seen.

Collectively, these results indicate that incorporation of FIAU into DNA may induce local conformational changes in DNA as has been shown for duplex DNA containing monofluoronucleotides ( 16 ). In the case of AP-1 binding to the TRE, the presence of FIAU, which contains an iodine in place of the 5-methyl group, may disrupt the contact normally occurring between key amino acid residues of the transcription factor with the 5-methyl groups of thymidines located adjacent to the center of dyad symmetry. The importance of such contacts was underscored by demonstrating that the substitution of the thymidines proximal to the center of dyad symmetry with 5-bromouracil led to extensive UV cross-linking with the Fos/Jun complex ( 27 ). In addition, the contribution of the 5-methyl groups in those thymidines which participate in the binding of the TRE to AP-1 has been demonstrated by replacing these thymidines with deoxyuridine and showing that binding of TRE to Fos/Jun (components of AP-1) is strongly reduced or even abolished ( 27 ). Furthermore, the crystal structure of GCN4 revealed that alanine-239 is in van der Waals contact with the 5-methyl groups of thymidines adjacent to the center of dyad symmetry of the TRE ( 29 ). Alanine-239 is absolutely conserved in the bZIP family of transcription factors which include c-fos and c-jun.

Effect of incorporated FIAU on restriction endonuclease digestion

In addition to affecting binding of AP-1 and TFIID, experiments were conducted to assess the effect of incorporated FIAU on the restriction endonuclease digestion of the oligonucleotide duplex. As shown in Table 3 , complete restriction was not seen with any oligomer (I-IV). The unsubstituted Oligomer IV was 70% digested with Mse I after 180 min. By comparison, no restriction was observed when the oligomer (Oligomer I) contained two FIAUs, one located on the 3'-side and the second on the 5'-side of the cleavage site. Restriction was limited to ~36% when FIAU was located on the 5' side of the cleavage site (Oligomer III). No effect on restriction was observed when FIAU was located only on the 3'-side of the cleavage site (Oligomer II).

Table 2 . Effect of FIAU on TFIID binding
Oligo code a

Response values for each replicate b

Least squares b

Rep. 1

Rep. 2

Rep. 3

Rep. 4

Rep. 5

Mean

Standard error

J

0.0488

0.0875

0.0758

0.0559

0.0390

0.0614

0.0112

K

0.0778

0.1407

0.1122

0.0854

0.0637

0.0960

0.0112

L

0.1058

0.1924

0.1806

0.1735

0.1149

0.1534 c

0.0112

S

0.0596

0.0960

0.1565

0.1396

0.0922

0.1088 c

0.0112

T

0.0270

0.0660

0.0367

0.0000

0.0105

0.0281

0.0112

U

0.0653

0.1122

0.0906

0.1082

0.0556

0.0864

0.0112

V

0.1652

0.2642

0.3286

0.2773

0.1987

0.2468 c

0.0112

X

- d

0.0264

0.0779

0.0719

0.0449

0.0553

0.0127

Y

0.0298

0.0804

0.0483

0.0465

0.0335

0.0477

0.0112

a The sequence of each oligonucleotide is shown in Figure 3A. b Values represent transformed (square root) response values and least squares mean and standard error for each oligoduplex as described in Materials and Methods. c Mean response is significantly greater than the mean response for oligo J by Dunnett's test. d No value was obtained for oligo duplex X in replicate 1.

Table 3 Effect of FIAU on restriction endonuclease digestion by Mse I or Bam HI
Oligomer duplex a

Sequence b

Restriction (%) c

I

5'-GAATG T*TAA CTAATGAGATC-3'

0

3'-CT TAC AAF*F GATTACTCTAG-5'

II

5'-GAATG T*TAA CTAATGAGATC-3'

69

3'-CT TAC AAF*T GATTACTCTAG-5'

III

5'-GAATG T*TAA CTAATGAGATC-3'

36

3'-CT TAC AAT*F GATTACTCTAG-5'

IV

5'-GAATG T*TAA CTAATGAGATC-3'

70

3'-CT TAC AAT*T GAT TACTCTAG-5'

V

5'-GATCTC ATTAG *GAFC CATTC-3'

68

3'-CATGAGTAATC CFAG* GTAAG-5'

VI

5'-GATCTC ATTAG* GAFCC ATTC-3'

81

3'-CATGAGTAATC CTAG* GTAAG-5'

VII

5'-GAATG *GAFC CTAATGAGATC-3'

86

3'-CTTAC CTAG* GATTACTCTAG-5'

VIII

5'-GATCTC ATTAG *GATC CATTC-3'

82

3'-CATGAGTAATC CTAG* GTAAG-5'

a Duplexes I-IV contain the recognition sequence for Mse I. Duplexes V-VIII contain the recognition sequence for Bam HI. b The bold letters in each sequence denote the restriction endonuclease recognition sequence. The asterisk indicates the cleavage site. F indicates the replacement of thymidine with FIAU. c The values in this column represent the extent of cutting of FIAU substituted (I-III and V-VII) and unsubstituted oligomers (IV and VIII) after 180 min. The extent of restriction endonuclease digestion in each case was determined as described in Materials and Methods.

These results demonstrate that the presence of FIAU 5' to the cleavage site in the consensus sequence T*TAA (where * is the cleavage site) inhibited restriction of the oligomeric duplex by Mse I. This decrease in the rate of cleavage by Mse I may be caused by the localized effects of the incorporation of FIAU adjacent to the phosphodiester bond of the cleavage site. As also shown in Table 3 , little or no effect on restriction cutting by Bam HI was observed when FIAU was located in the consensus sequence *GATC. However, the presence of two FIAUs in the restriction site, one in each DNA strand, did appear to have a slight effect on enzyme digestion (68% digestion of Oligomer V digestion compared with 82% digestion of unsubstituted Oligomer VIII). The effects of various nucleoside analogs on restriction enzyme activity are well documented ( 31 - 33 ) and these results are reminiscent of results observed with difluorodeoxycytidine ( 17 ). Additional research will be required to determine whether such effects are operative on DNA modifying enzymes present in mammalian cells.

The results reported here indicate that incorporation of FIAU into DNA leads to localized changes rather than global disruptions in DNA topography. These localized topographical changes may lead to the disruption of DNA replication and/or normal gene expression by inhibiting or enhancing essential DNA-protein interactions. Consequently, the antiviral activity and/or cytotoxicity of FIAU may be due, in part, to the ability of this compound to alter DNA-protein contacts and hence modulate viral and cellular gene expression.

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*To whom correspondence should be addressed. Tel: +1 317 276-4288; Fax: +1 317 276-1743; Email: colacino_joseph@lilly.com
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