title: In vitro and in vivo dimerization of human endonuclease III stimulates its activity.
abstract: Human endonuclease III (hNTH1), a DNA glycosylase with associated abasic lyase activity, repairs various mutagenic and toxic-oxidized DNA lesions, including thymine glycol. We demonstrate for the first time that the full-length hNTH1 positively cooperates in product formation as a function of enzyme concentration. The protein concentrations that caused cooperativity in turnover also exhibited dimerization, independent of DNA binding. Earlier we had found that the hNTH1 consists of two domains: a well conserved catalytic domain, and an inhibitory N-terminal tail. The N-terminal truncated proteins neither undergo dimerization, nor do they show cooperativity in turnover, indicating that the homodimerization of hNTH1 is specific and requires the N-terminal tail. Further kinetic analysis at transition states reveals that this homodimerization stimulates an 11-fold increase in the rate of release of the final product, an AP-site with a 3'-nick, and that it does not affect other intermediate reaction rates, including those of DNA N-glycosylase or AP lyase activities that are modulated by previously reported interacting proteins, YB-1, APE1, and XPG. Thus, the site of modulating action of the dimer on the hNTH1 reaction steps is unique. Moreover, the high intranuclear (2.3 microM) and cytosolic (0.65 microM) concentrations of hNTH1 determined here support the possibility of in vivo dimerization; indeed, in vivo protein cross-linking showed the presence of the dimer in the nucleus of HeLa cells. Therefore, it is likely that the dimerization of hNTH1 involving the N-terminal tail masks the inhibitory effect of this tail and plays a critical role in its catalytic turnover in the cell.
authors.affiliation: DNA Repair Laboratory, Mechanism of Carcinogenesis Program, American Health Foundation Cancer Center, Institute for Cancer Prevention, Valhalla, New York 10595, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Liu
authors.forename: Sujata
authors.initials: S
authors.lastname: Choudhury
authors.forename: Rabindra
authors.initials: R
authors.lastname: Roy
mesh_terms: D000626:Aminopeptidases / Q000378:metabolism; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002467:Cell Nucleus / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D045647:DNA Glycosylases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005557:Formaldehyde / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; D000099250:DNA Excision Repair Protein ERCC-5
chemical_list: D000626:Aminopeptidases; D022762:CCAAT-Enhancer-Binding Proteins; D003432:Cross-Linking Reagents; D004247:DNA; D045647:DNA Glycosylases; D004268:DNA-Binding Proteins; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004720:Endonucleases; D029968:Escherichia coli Proteins; D005557:Formaldehyde; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; D000099250:DNA Excision Repair Protein ERCC-5; C070856:YBX1 protein, human; C106403:NTH protein, E coli; C019689:APE1 protein, S cerevisiae