DNA end processing by polynucleotide kinase/phosphatase.

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, United States Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 12/2011; 108(52):20855-6. DOI: 10.1073/pnas.1118214109
Source: PubMed
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    ABSTRACT: The topoisomerase II (topo II) DNA incision-and-ligation cycle can be poisoned (for example following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double-strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5'-phosphotyrosyl-linked topo II-DNA adducts. Here, X-ray structures of mouse Tdp2-DNA complexes reveal that Tdp2 β-2-helix-β DNA damage-binding 'grasp', helical 'cap' and DNA lesion-binding elements fuse to form an elongated protein-DNA conjugate substrate-interaction groove. The Tdp2 DNA-binding surface is highly tailored for engagement of 5'-adducted single-stranded DNA ends and restricts nonspecific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analyses support a single-metal ion catalytic mechanism for the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily and establish a molecular framework for targeted small-molecule blockade of Tdp2-mediated resistance to anticancer topoisomerase drugs.
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    ABSTRACT: Phosphorylation of DNA by polynucleotide kinase (PNK) takes an important role in DNA damage repair, replication and recombination. The evaluation of PNK activity has received an increasing attention due to the significance of PNK. Here, we present a label-free fluorescent method for PNK activity assay using double strand DNA (dsDNA) -templated copper nanoparticles (CuNPs) as a fluorescent indicator. Upon the PNK reaction, the dsDNA template is phosphorylated and then digested by λ exonuclease immediately, prohibiting the formation of fluorescent CuNPs due to the lack of dsDNA template. This homogeneous PNK activity assay does not require any other additional modifications of DNA substrate or complex design, making the proposed strategy simple, cost-effective and high throughput. The proposed strategy is sensitive, selective and exhibits a good assay performance in complex biological samples. The strategy presented here opens a new avenue for PNK assay and nucleic acid phosphorylation related research.
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