Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells

Ranajoy Chattopadhyay; Lee Wiederhold; Bartosz Szczesny; Istvan Boldogh; Tapas K. Hazra; Tadahide Izumi; Sankar Mitra

doi:10.1093/nar/gkl177

Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells

Ranajoy Chattopadhyay, Lee Wiederhold, Bartosz Szczesny, Istvan Boldogh, Tapas K. Hazra, Tadahide Izumi, Sankar Mitra

Toxicology and Cancer Biology

Research output: Contribution to journal › Article › peer-review

130 Scopus citations

Abstract

Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3′ blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35 S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.

Original language	English
Pages (from-to)	2067-2076
Number of pages	10
Journal	Nucleic Acids Research
Volume	34
Issue number	7
DOIs	https://doi.org/10.1093/nar/gkl177
State	Published - 2006

Bibliographical note

Funding Information:
We acknowledge Dr R. Roy’s initial studies in purification of mitochondria and characterization of APE1 antibody cross reacting proteins. We also thank Dr David Konkel for critical editing of this manuscript, and Ms. Wanda Smith for expert secretarial assistance. We are grateful to A. Kurosky and S. S. Smith of the Biomolecular Resource Facility for protein characterization. This work was supported by USPHS grants R01 CA53791, ES08457, P01 AG021830 (S.M.), CA98664 (T.I.), and NIEHS Center Grant ES06676. Funding to pay the Open Access publication charges for this article was provided by R01 CA53791.

Funding

We acknowledge Dr R. Roy’s initial studies in purification of mitochondria and characterization of APE1 antibody cross reacting proteins. We also thank Dr David Konkel for critical editing of this manuscript, and Ms. Wanda Smith for expert secretarial assistance. We are grateful to A. Kurosky and S. S. Smith of the Biomolecular Resource Facility for protein characterization. This work was supported by USPHS grants R01 CA53791, ES08457, P01 AG021830 (S.M.), CA98664 (T.I.), and NIEHS Center Grant ES06676. Funding to pay the Open Access publication charges for this article was provided by R01 CA53791.

Funders	Funder number
National Institute of Environmental Health Sciences (NIEHS)	R01ES008457, ES06676
U.S. Public Health Service	R01 CA53791, P01 AG021830, CA98664, ES08457

ASJC Scopus subject areas

Genetics

Access to Document

10.1093/nar/gkl177

Cite this

@article{c9e9efa4ae2744508fe7eb40da7e9877,

title = "Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells",

abstract = "Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3′ blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35 S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.",

author = "Ranajoy Chattopadhyay and Lee Wiederhold and Bartosz Szczesny and Istvan Boldogh and Hazra, {Tapas K.} and Tadahide Izumi and Sankar Mitra",

note = "Funding Information: We acknowledge Dr R. Roy{\textquoteright}s initial studies in purification of mitochondria and characterization of APE1 antibody cross reacting proteins. We also thank Dr David Konkel for critical editing of this manuscript, and Ms. Wanda Smith for expert secretarial assistance. We are grateful to A. Kurosky and S. S. Smith of the Biomolecular Resource Facility for protein characterization. This work was supported by USPHS grants R01 CA53791, ES08457, P01 AG021830 (S.M.), CA98664 (T.I.), and NIEHS Center Grant ES06676. Funding to pay the Open Access publication charges for this article was provided by R01 CA53791.",

year = "2006",

doi = "10.1093/nar/gkl177",

language = "English",

volume = "34",

pages = "2067--2076",

number = "7",

}

TY - JOUR

T1 - Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells

AU - Chattopadhyay, Ranajoy

AU - Wiederhold, Lee

AU - Szczesny, Bartosz

AU - Boldogh, Istvan

AU - Hazra, Tapas K.

AU - Izumi, Tadahide

AU - Mitra, Sankar

N1 - Funding Information: We acknowledge Dr R. Roy’s initial studies in purification of mitochondria and characterization of APE1 antibody cross reacting proteins. We also thank Dr David Konkel for critical editing of this manuscript, and Ms. Wanda Smith for expert secretarial assistance. We are grateful to A. Kurosky and S. S. Smith of the Biomolecular Resource Facility for protein characterization. This work was supported by USPHS grants R01 CA53791, ES08457, P01 AG021830 (S.M.), CA98664 (T.I.), and NIEHS Center Grant ES06676. Funding to pay the Open Access publication charges for this article was provided by R01 CA53791.

PY - 2006

Y1 - 2006

N2 - Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3′ blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35 S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.

AB - Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3′ blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35 S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.

UR - http://www.scopus.com/inward/record.url?scp=33645928711&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33645928711&partnerID=8YFLogxK

U2 - 10.1093/nar/gkl177

DO - 10.1093/nar/gkl177

M3 - Article

C2 - 16617147

AN - SCOPUS:33645928711

SN - 0305-1048

VL - 34

SP - 2067

EP - 2076

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 7

ER -

Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells

Abstract

Bibliographical note

Funding

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this