title: Truncation of amino-terminal tail stimulates activity of human endonuclease III (hNTH1).
abstract: The human base excision repair enzyme hNTH1, a homologue of Escherichia coli endonuclease III (Nth), is a 36kDa DNA glycosylase with associated abasic (AP) lyase activity. It has significant sequence homology with Nth in its DNA-binding motifs and catalytic residues but possesses a unique amino (N)-terminal tail (residues 1-95). We investigated the structure and function of this tail. Controlled proteolysis cleaved hNTH1 into discrete fragments to generate a 25kDA core domain lacking the N-terminal 98 residues. Surprisingly, recombinant hNTH1 lacking 55, 72 or 80 residues from the N terminus had four- to fivefold higher activities than the full-length enzyme. Kinetic analysis at transition states revealed that release of the final product, an AP site with a 3'-nick, is the rate-limiting step in the multi-step reaction mediated by hNTH1. The N-terminal tail regulates its overall catalytic turnover by reducing this product release rate by five- to sevenfold without affecting either the glycosylase or AP lyase activities, or the steady-state equilibrium concentration of Schiff base intermediate, the covalent complex of hNTH1 and AP-site DNA formed after the base is excised. The inhibitory role of the N-terminal tail in catalytic turnover explains the low activity of hNTH1 compared to that of its E.coli homologue.
authors.affiliation: Division of Carcinogenesis and Molecular Epidemiology, American Health Foundation Cancer Center, 1 Dana Road, Valhalla, NY 10595, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Liu
authors.forename: Rabindra
authors.initials: R
authors.lastname: Roy
mesh_terms: D000595:Amino Acid Sequence; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D045647:DNA Glycosylases; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012545:Schiff Bases / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics*; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D012545:Schiff Bases; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; C468347:NTHL1 protein, human; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D014357:Trypsin; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase