Abstract
Human endonuclease III (hNth1) is a DNA glycosylase/apurinic/apyrimidinic (AP) lyase that initiates base excision repair of pyrimidines modified by reactive oxygen species, ionizing, and ultraviolet radiation. Using duplex 2'-deoxyribose oligonucleotides containing an abasic (AP) site, a thymine glycol, or a 5-hydroxyuracil residue as substrates, we found the AP lyase activity of hNth1 was 7 times slower than its DNA glycosylase activity, similar to results reported for murine and human 8-oxoguanine-DNA glycosylase, which are also members of the endonuclease III family. This difference in rates contrasts with the equality of rates found in Escherichia coli and Saccharomyces cerevisiae endonuclease III homologs. A yeast two-hybrid screen for potential modulators of hNth1 activity revealed interaction with the damage-inducible transcription factor Y box-binding protein 1 (YB-1), also identified 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. Analysis revealed that YB-1 affects the steady state equilibrium between the covalent hNth1-AP site Schiff base ES intermediate and the noncovalent ES intermediate containing the AP aldehydic sugar and the epsilon-amino group of the hNth1 active site lysine. This equilibrium may be a checkpoint in modulating hNth1 activity.
Highlights
Human endonuclease III is a DNA glycosylase/ apurinic/apyrimidinic (AP) lyase that initiates base excision repair of pyrimidines modified by reactive oxygen species, ionizing, and ultraviolet radiation
Using duplex 2-deoxyribose oligonucleotides containing an abasic (AP) site, a thymine glycol, or a 5-hydroxyuracil residue as substrates, we found the AP lyase activity of hNth1 was 7 times slower than its DNA glycosylase activity, similar to results reported for murine and human 8-oxoguanine-DNA glycosylase, which are members of the endonuclease III family
His6YB-1 Stimulates hNth1 Activity against High Molecular Weight Substrates Containing AP Sites, thymine glycol (Tg), or Cytosine Hydrate Residues—To determine whether there was an effect of Y box-binding protein 1 (YB-1) on hNth1-mediated catalysis, the activity of a fixed amount of hNth1 was assayed in the presence of varying concentrations of YB-1 using three different high molecular weight substrates: supercoiled plasmid DNA containing AP sites created by acid hydrolysis of purines; poly(dA-[3H]dT)1⁄7poly(dA[3H]dT) containing multiple Tg residues formed by oxidation with osmium tetroxide; and poly(dG-[3H]dC)1⁄7poly(dG-[3H]dC) containing multiple cytosine hydrate residues formed by ultraviolet irradiation
Summary
Using duplex 2-deoxyribose oligonucleotides containing an abasic (AP) site, a thymine glycol, or a 5-hydroxyuracil residue as substrates, we found the AP lyase activity of hNth was 7 times slower than its DNA glycosylase activity, similar to results reported for murine and human 8-oxoguanine-DNA glycosylase, which are members of the endonuclease III family. Multiple turnover assays were performed individually in 20-l volumes, containing reaction buffer, 100 nM 2Ј-deoxyribose oligonucleotide substrate duplex, and 10 nM hNth with or without 10 nM His6YB-1. Enzyme nicking assays were performed in the HEPES reaction buffer listed above in a volume of 10 l, containing 500 –750 ng of treated pBR322 DNA and 5 nM hNth with or without 10 nM His6YB-1. The bands were visualized by ethidium bromide staining and quantitated by densitometry using the Molecular Imager FX System with Quantity One Software (Bio-Rad)
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