Human thymine DNA glycosylase binds to apurinic sites in DNA but is displaced by human apurinic endonuclease 1.
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.
Waters
T R
TR
Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom. t.waters@biochem.ucl.ac.uk
Gallinari
P
P
Jiricny
J
J
Swann
P F
PF
D020137:Base Pair Mismatch; D001483:Base Sequence; D001667:Binding, Competitive; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D002384:Catalysis; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding
D017931:DNA Primers; D004247:DNA; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D043224:Deoxyribonuclease (Pyrimidine Dimer); D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase