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Nucleic Acids Research, 2001, Vol. 29, No. 11 2349-2360
© 2001 Oxford University Press

Human APE2 protein is mostly localized in the nuclei and to some extent in the mitochondria, while nuclear APE2 is partly associated with proliferating cell nuclear antigen

Daisuke Tsuchimoto1,2, Yasunari Sakai1,2, Kunihiko Sakumi1,2, Kenichi Nishioka1,2, Masafumi Sasaki3, Toshiyuki Fujiwara4 and Yusaku Nakabeppu1,2,*

1Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan, 2CREST, Japan Science and Technology Corporation, Japan, 3Department of Virology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan and 4Department of Biochemistry, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan

In human cells APE1 is the major AP endonuclease and it has been reported to have no functional mitochondrial targeting sequence (MTS). We found that APE2 protein possesses a putative MTS. When its N-terminal 15 amino acid residues were fused to the N-terminus of green fluorescent protein and transiently expressed in HeLa cells the fusion protein was localized in the mitochondria. By electron microscopic immunocytochemistry we detected authentic APE2 protein in mitochondria from HeLa cells. Western blotting of the subcellular fraction of HeLa cells revealed most of the APE2 protein to be localized in the nuclei. We found a putative proliferating cell nuclear antigen (PCNA)-binding motif in the C-terminal region of APE2 and showed this motif to be functional by immunoprecipitation and in vitro pull-down binding assays. Laser scanning immunofluorescence microscopy of HeLa cells demonstrated both APE2 and PCNA to form foci in the nucleus and also to be co-localized in some of the foci. The incubation of HeLa cells in HAT medium containing deoxyuridine significantly increased the number of foci in which both molecules were co-localized. Our results suggest that APE2 participates in both nuclear and mitochondrial BER and also that nuclear APE2 functions in the PCNA-dependent BER pathway.

* To whom correspondence should be addressed at: Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka 812-8582, Japan. Tel: +81 92 642 6800; Fax: +81 92 642 6791; Email: yusaku{at}bioreg.kyushu-u.ac.jp


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