Stimulation of human endonuclease III by Y box-binding protein 1 (DNA-binding protein B) - Interaction between a base excision repair enzyme and a transcription factor

Human endonuclease III (hNth1) is a DNA glycosylase/ apurinic/apyrimidinic (AP) lyase that initiates base excision repair of pyrimidines modified by r eactive oxygen species, ionizing, and ultraviolet radiation. Using duplex 2 '-deoxyribose oligonucleotides containing an abasic (AP) site, a thymine gl ycol, or a 5-hydroxyuracil residue as substrates, we found the AP lyase act ivity of hNth1 was 7 times slower than its DNA glycosylase activity, simila r to results reported for murine and human 8-oxoguanine-DNA glycosylase, wh ich are also members of the endonuclease III family. This difference in rat es contrasts with the equality of rates found in Escherichia coil and Sacch aromyces cerevisiae endonuclease III homologs, A yeast two-hybrid screen fo r potential modulators of hNth1 activity revealed interaction with the dama ge-inducible transcription factor Y box-binding protein 1 (YB-1), also iden tified as DNA-binding protein B (DbpB). The in vitro addition of His(6)YB-1 to hNth1 increased the rate of DNA glycosylase and AP lyase activity. Anal ysis revealed that YB-1 affects the steady state equilibrium between the co valent hNth1-AP site Schiff base ES intermediate and the noncovalent ES int ermediate containing the AP aldehydic sugar and the epsilon -amino group of the hNth1 active site lysine, This equilibrium may be a checkpoint in modu lating hNth1 activity.