Abstract
In vitro, following the removal of thymine from a G.T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed. It can also bind to an apurinic site opposite S6-methylthioguanine (SMeG) or opposite any of the remaining natural DNA bases. It will therefore bind to apurinic sites formed by spontaneous depurination, chemical attack, or other glycosylases. In the absence of magnesium, the rate of dissociation of the glycosylase from such complexes is so slow (koff 1.8 - 3.6 x 10(-5) s-1; i.e. half-life between 5 and 10 h) that each molecule of glycosylase removes essentially only one molecule of thymine. In the presence of magnesium, the dissociation rates of the complexes with C.AP and SMeG.AP are increased more than 20-fold, allowing each thymine DNA glycosylase to remove more than one uracil or thymine from C.U and SMeG.T mismatches in DNA. In contrast, magnesium does not increase the dissociation of thymine DNA glycosylase from G.AP sites sufficiently to allow it to remove more than one thymine from G.T mismatches. The bound thymine DNA glycosylase prevents human apurinic endonuclease 1 (HAP1) cutting the apurinic site, so unless the glycosylase was displaced, the repair of apurinic sites would be very slow. However, HAP1 significantly increases the rate of dissociation of thymine DNA glycosylase from apurinic sites, presumably through direct interaction with the bound glycosylase. This effect is concentration-dependent and at the probable normal concentration of HAP1 in cells the dissociation would be fast. This interaction couples the first step in base excision repair, the glycosylase, to the second step, the apurinic endonuclease. The other proteins involved in base excision repair, polymerase beta, XRCC1, and DNA ligase III, do not affect the dissociation of thymine DNA glycosylase from the apurinic site.
Highlights
In vitro, following the removal of thymine from a G1⁄7T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed
Formation of the complex does not depend upon the apurinic site being opposite a guanine, since the glycosylase bound to DNA containing an apurinic site opposite S6-methylthioguanine [7], thymine (Fig. 2B), cytosine (Fig. 2C), or adenine (Fig. 2D)
Complete In Vitro Repair of G1⁄7T Mismatches—The experiments reported above show that thymine DNA glycosylase binds strongly to the apurinic sites produced from its supposed physiological substrate, a G1⁄7T mismatch in the sequence CpG1⁄7T, but that the thymine DNA glycosylase can be displaced from this complex by human apurinic endonuclease 1 (HAP1), the enzyme that catalyzes the second step in base excision repair
Summary
Enzymes—Thymine DNA glycosylase was expressed in Escherichia coli as described previously [8] and was purified in four chromatographic steps [7]. Experiments involving polymerase , XRCC1, and DNA ligase III were carried out in magnesium reaction buffer containing 20 M dCTP In these experiments, all proteins (including thymine DNA glycosylase and HAP1) were preincubated for 12 min at room temperature before adding the DNA containing a G1⁄7T mismatch. Samples were removed from this master mix at various times and added to 100-fold excess of unlabeled G1⁄7AP DNA, and incubation was continued at room temperature These samples were all loaded at the same time onto a non-denaturing polyacrylamide gel, and electrophoresis was carried out as described for the band shift experiments. The amount of bound 32P-labeled DNA was plotted against the time between adding the unlabeled competitor G1⁄7AP DNA and loading the sample onto the gel
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