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J. Biol. Chem., Vol. 278, Issue 50, 50061-50069, December 12, 2003

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In Vitro and in Vivo Dimerization of Human Endonuclease III Stimulates Its Activity^*

Xiang Liu, Sujata Choudhury, and Rabindra Roy¶

From the DNA Repair Laboratory, Mechanism of Carcinogenesis Program, American Health Foundation Cancer Center, Institute for Cancer Prevention, Valhalla, New York 10595

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 µM) and cytosolic (0.65 µM) 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.

Received for publication, September 8, 2003 , and in revised form, September 24, 2003.

^* This work was supported in part by National Institutes of Health RO1 Grant CA80917 and NCI National Institutes of Health Cancer Center Grant P30 CA17613. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Pharmacology, SUNY at Stony Brook, Stony Brook, NY 11794.

Both authors contributed equally to this work.

^¶ To whom correspondence should be addressed: DNA Repair Laboratory, American Health Foundation Cancer Center, Institute For Cancer Prevention, 1 Dana Rd., Valhalla, NY 10595. Tel.: 914-789-7130; Fax: 914-592-6317; E-mail: rroy{at}ifcp.us.

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