Human Thymine DNA Glycosylase Binds to Apurinic Sites in DNA but Is Displaced by Human Apurinic Endonuclease 1

Abstract

In vitro, following the removal of thymine from a G⅐T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed. It can also bind to an apurinic site opposite S 6 -methylthioguanine ( SMe G) 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 (k off 1.8 ؊ 3.6 ؋ 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 C⅐AP and SMe G⅐AP are increased more than 20-fold, allowing each thymine DNA glycosylase to remove more than one uracil or thymine from C⅐U and SMe G⅐T mismatches in DNA. In contrast, magnesium does not increase the dissociation of thymine DNA glycosylase from G⅐AP sites sufficiently to allow it to remove more than one thymine from G⅐T 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 ␤, XRCC1, and DNA ligase III, do not affect the dissociation of thymine DNA glycosylase from the apurinic site.In mammalian cells, 2-7% of the total cytosine is methylated. Spontaneous deamination of 5-methylcytosine, which is somewhat faster than cytosine (1), generates G⅐T mispairs in DNA. The repair of these G⅐T mismatches is initiated by thymine DNA glycosylase which excises the mismatched thymine (2, 3). 5-Methylcytosine occurs almost exclusively in the sequence Me CpG, and in keeping with its proposed role in the repair of G⅐T mispairs resulting from the deamination of 5-methylcytosine, thymine DNA glycosylase shows a strong preference for removal of thymine from CpG⅐T sequences (4 -7). The human enzyme has been cloned and overexpressed (8) and has been shown to belong to a family of uracil DNA glycosylases that remove uracil from G⅐U base pairs but that are distinct from the general uracil DNA glycosylase enzyme (9). Thymine DNA glycosylase removes uracil from G⅐U base pairs more rapidly than it removes thymine from G⅐T base pairs (10) and can also remove uracil from C⅐U, T⅐U, and A⅐U base pairs (7) and may therefore provide a backup function to the general uracil DNA glycosylase. The glycosylase also removes thymine from base pairs with S 6 -methylthioguanine ( SMe G) 1 that ar...

Uracil in DNA – occurrence, consequences and repair

“…Therefore, APE1 most likely displaces TDG because it has even higher affinity for the AP-site than TDG, and in addition APE1 is more abundant. In agreement with this, APE1 strongly stimulates the activity of TDG (Waters et al, 1999). Recent findings indicate, however, that the AP-site binding capacity of TDG might be subject to regulation.…”

“…Such rebinding has subsequently been observed for several DNA glycosylases (Vidal et al, 2001;Waters et al, 1999). Recent studies have, however, demonstrated that the human nuclear UNG2 does not bind AP-sites (Kavli et al, 2002), and the in vivo significance of APsite shielding thus remains elusive, at least for UNG2.…”

Uracil in DNA and its processing by different DNA glycosylases

“…TDG is an intriguing protein that, similar to SMUG1, has a low turnover number and strong binding to AP sites, and its activity is stimulated by APE1. As with SMUG1, the binding of the glycosylase to the AP site inhibits cleavage by the downstream AP endonuclease ( Waters et al 1999). Interestingly, the catalytic efficiency of the protein is increased by SUMOylation (Hardeland et al 2002).…”

Vpr-mediated Incorporation of UNG2 into HIV-1 Particles Is Required to Modulate the Virus Mutation Rate and for Replication in Macrophages

“…Uracil-DNA glycosylase inhibitor was obtained from New England Biolabs (Beverly, MA). Assays of UNG activity were done with the single-stranded DNA oligonucleotide substrate and were performed in UNG reaction buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, pH 8.0) at 37°C for 1 h. Apurinic sites were cleaved by adding 1 ⁄2 volume of 0.5 M NaOH and 1 ⁄2 volume of 30 mM EDTA and then boiling for 30 min (27). Samples were then applied to a nondenaturing 20% polyacrylamide gel with electrophoresis at 60 V for 3.5 h or were applied to a denaturing 19% polyacrylamide gel run at 400 V for 2 h. Gels were stained with SYBR Gold (Molecular Probes, Eugene, OR), and nucleic acids were visualized with an ultraviolet transilluminator.…”