An anthraquinone-linked duplex DNA oligomer containing 60 base pairs was synthesized by PCR. The strand complementary to the quinone-containing strand has four isolated GG steps, which serve as traps for a migrating radical cation. Irradiation of the quinone leads to electron transfer from the DNA to the quinone forming the anthraquinone radical anion and a base radical cation. The radical cation migrates through the DNA, causing reaction at GG steps revealed as strand breaks. The efficiency of strand cleavage falls off exponentially with distance from the quinone (slope ؍ ؊0.02 Å ؊1 ). This finding necessitates reinterpretation of mechanisms proposed for radical cation migration in DNA. We propose that radical cations form self-trapped polarons that migrate by thermally activated hopping.
A series of cationic anthraquinone derivatives was investigated for their ability to stabilize duplex and triplex DNA. Thermal denaturation experiments demonstrate that each of these compounds stabilizes the [poly(dT) x poly(dA) x poly(dT)] triplex without significantly affecting the [poly(dT) x poly(dA)] duplex. The amount of stabilization is determined by the number and placement of the cationic substituents on the anthraquinone skeleton. The stabilization arises primarily from higher affinity binding of the quinones to the triplex relative to the duplex structures. Phosphorescence quenching and viscometric titrations indicate that the quinones bind to the triplex by intercalation.
Peptide nucleic acids (PNA) are mimics with normal bases connected to a pseudopeptide chain that obey Watson-Crick rules to form stable duplexes with itself and natural nucleic acids. This has focused attention on PNA as therapeutic or diagnostic reagents. Duplexes formed with PNA mirror some but not all properties of DNA. One fascinating aspect of PNA biochemistry is their reaction with enzymes. Here we show an enzyme reaction that operates effectively on a PNA͞DNA hybrid duplex. A DNA oligonucleotide containing a cis, syn-thymine [2؉2] dimer forms a stable duplex with PNA. The hybrid duplex is recognized by photolyase, and irradiation of the complex leads to the repair of the thymine dimer. This finding provides insight into the enzyme mechanism and provides a means for the selective repair of thymine photodimers.UV light damages nucleic acids primarily by causing dimerization of adjacent pyrimidines. This damage is repaired enzymatically by DNA photolyase, which binds selectively to cis, syn-cyclobutane dimers and, when this complex is activated by light, reforms the monomeric pyrimidines (1, 2). These reactions are outlined in Scheme 1 for thymines. Thymine photodimerization and its repair is an appealing system for examination of parallels in the properties of peptide nucleic acids (PNA), Scheme 2, and DNA. Photolyase is a siteselective, rather than a sequence-selective, enzyme. It binds to thymine dimers whether they are incorporated in superhelical, circular, linear, or single-stranded DNA. It appears that the only additional requirement for recognition by the enzyme is phosphate groups on the dimer-containing strand (3, 4). For these reasons, we suspected that photolyase might retain its ability to recognize and repair thymine dimers contained on the DNA strand of a DNA͞PNA hybrid duplex (5-8). We carried out a series of experiments to assess this possibility.
MATERIALS AND METHODSMelting Temperature (T m ) Measurements. Absorbance versus temperature curves were measured at 260 nm in 10 mM phosphate buffer at pH 7 for solutions containing 2 M of each strand of PNA or DNA shown in Scheme 3. The melting temperature, T m , is assigned as the peak of the first derivative plot.Gel Mobility Assay. The oligonucleotides DNA(1) and DNA(3) were labeled with 32 P at the 5Ј end by using standard techniques (9). Separate samples of the radiolabeled DNA (2,500 cpm) were mixed with complementary DNA(2) (5 M), PNA(1), or PNA(4) (5 M) in 10 l of 10 mM phosphate buffer at pH 7. The hybridization was carried out by heating to 90°C for 5 min and then cooling the solution to room temperature for 2 h. The samples were dried with a Speedvac at low heat for 2 h, and 5 l of nondenaturing loading buffer was added. The analysis was carried out by electrophoresis in a 20% polyacrylamide gel (29:1 acrylamide and bisacrylamide) followed by autoradiography. The gel was run with TBE buffer containing 89 mM Tris-borate and 1 mM EDTA, pH 8.
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