Exploring DNA destabilization induced by the thymine dimer lesion using base
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Date
2011-02-16
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Abstract
DNA frequently forms mutations due to endogenous or environmental
conditions. During the Base Excision Repair pathway (BER), an enzyme
removes damaged bases from DNA with a series of enzymatic steps. The
thymine dimer lesion is formed from the cycloaddition of two same-strand,
neighboring thymines. A kink in the duplex DNA occurs because the thymine dimer lesion cannot form hydrogen bonds with complementary adenines. Despite the extensive research done on the thymine dimer mutation and its repair mechanism, little is known about the way in which the specific BER enzyme locates the DNA lesion in order to carry out the BER pathway. It is possible that the thymine dimer lesion causes thermodynamic and kinetic destabilization to the DNA duplex.
In order to study DNA destabilization caused by the thymine dimer, DNA containing the dimer was purified and labeled with the radioactive isotope, 32P. The DNA was reacted with DNA base modifying chemical probes, Dimethyl Sulfate (DMS), Potassium Permanganate (KMnO4), and Diethyl pyrocarbonate
(DEPC). Following the base modification, the sugar phosphate backbone of the DNA was cleaved using piperidine. The fragments were observed using electrophoretic techniques. Data from this method has revealed significant reactivity of the bases around the thymine dimer. This suggests that the thymine
dimer lesion kinetically destabilizes surrounding bases in duplex DNA.
A standard method for measuring destabilization induced by a DNA lesion involves thermodynamic techniques and optical measurements. A temperature-controlled UV-vis spectrophotometer was used to observe differences in the shape
of the sigmoidal melting curves and thermodynamic parameters. Differential Scanning Calorimetry (DSC) was used to determine and confirm values obtained through spectrophotometry. The thermal melting of the B-form duplex strand
contrasts with an enthalpically and thermally destabilizing thymine dimer duplex.
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Keywords
Thymine, Dimer, DNA, destabilization