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Phosphorylation of polynucleotide kinase/ phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage.


ABSTRACT: Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5'-DNA kinase/3'-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.

SUBMITTER: Zolner AE 

PROVIDER: S-EPMC3241656 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Phosphorylation of polynucleotide kinase/ phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage.

Zolner Angela E AE   Abdou Ismail I   Ye Ruiqiong R   Mani Rajam S RS   Fanta Mesfin M   Yu Yaping Y   Douglas Pauline P   Tahbaz Nasser N   Fang Shujuan S   Dobbs Tracey T   Wang Chen C   Morrice Nick N   Hendzel Michael J MJ   Weinfeld Michael M   Lees-Miller Susan P SP  

Nucleic acids research 20110808 21


Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5'-DNA kinase/3'-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was se  ...[more]

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