Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3′-phosphatase in maintenance of mitochondrial genome

Santi M. Mandal, Muralidhar L. Hegde, Arpita Chatterjee, Pavana M. Hegde, Bartosz Szczesny, Dibyendu Banerjee, Istvan Boldogh, Rui Gao, Maria Falkenberg, Claes M. Gustafsson, Partha Sarkar, Tapas Hazra

Research output: Contribution to journalArticle

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Abstract

The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3′-phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion- specific DNA polymerase γ. In cell association of NEIL2 and PNKP with polymerase γ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3′-phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.

Original languageEnglish (US)
Pages (from-to)2819-2829
Number of pages11
JournalJournal of Biological Chemistry
Volume287
Issue number4
DOIs
StatePublished - Jan 20 2012

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Polynucleotide 5'-Hydroxyl-Kinase
DNA Glycosylases
Mitochondrial Genome
Phosphoric Monoester Hydrolases
Protein Kinases
Repair
Genes
Maintenance
Proteins
Mitochondria
Electron Transport Complex IV
Association reactions
Mitochondrial Genes
Chromatin Immunoprecipitation
HEK293 Cells
DNA-Directed DNA Polymerase
Enzymes
Confocal microscopy
Confocal Microscopy
Ligation

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3′-phosphatase in maintenance of mitochondrial genome. / Mandal, Santi M.; Hegde, Muralidhar L.; Chatterjee, Arpita; Hegde, Pavana M.; Szczesny, Bartosz; Banerjee, Dibyendu; Boldogh, Istvan; Gao, Rui; Falkenberg, Maria; Gustafsson, Claes M.; Sarkar, Partha; Hazra, Tapas.

In: Journal of Biological Chemistry, Vol. 287, No. 4, 20.01.2012, p. 2819-2829.

Research output: Contribution to journalArticle

Mandal, Santi M. ; Hegde, Muralidhar L. ; Chatterjee, Arpita ; Hegde, Pavana M. ; Szczesny, Bartosz ; Banerjee, Dibyendu ; Boldogh, Istvan ; Gao, Rui ; Falkenberg, Maria ; Gustafsson, Claes M. ; Sarkar, Partha ; Hazra, Tapas. / Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3′-phosphatase in maintenance of mitochondrial genome. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 4. pp. 2819-2829.
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T1 - Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3′-phosphatase in maintenance of mitochondrial genome

AU - Mandal, Santi M.

AU - Hegde, Muralidhar L.

AU - Chatterjee, Arpita

AU - Hegde, Pavana M.

AU - Szczesny, Bartosz

AU - Banerjee, Dibyendu

AU - Boldogh, Istvan

AU - Gao, Rui

AU - Falkenberg, Maria

AU - Gustafsson, Claes M.

AU - Sarkar, Partha

AU - Hazra, Tapas

PY - 2012/1/20

Y1 - 2012/1/20

N2 - The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3′-phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion- specific DNA polymerase γ. In cell association of NEIL2 and PNKP with polymerase γ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3′-phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.

AB - The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3′-phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion- specific DNA polymerase γ. In cell association of NEIL2 and PNKP with polymerase γ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3′-phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.

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