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PLoS One. 2010 Aug 17;5(8):e12241. doi: 10.1371/journal.pone.0012241.

Genetic and biochemical characterization of human AP endonuclease 1 mutants deficient in nucleotide incision repair activity.

Author information

1
CNRS UMR8126, Université Paris-Sud, Institut de Cancérologie Gustave Roussy, Villejuif, France.

Abstract

BACKGROUND:

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5' to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.

METHODOLOGY/PRINCIPAL FINDING:

We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3'-->5' exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase beta and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.

CONCLUSION/SIGNIFICANCE:

Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.

PMID:
20808930
PMCID:
PMC2923195
DOI:
10.1371/journal.pone.0012241
[Indexed for MEDLINE]
Free PMC Article

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