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Chemical shift assignments for human apurinic/apyrimidinic endonuclease 1

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Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1 or Ref-1) is the major enzyme in mammals for processing abasic sites in DNA. These cytotoxic and mutagenic lesions arise via spontaneous rupture of the base-sugar bond or the removal of damaged bases by a DNA glycosylase. APE1 cleaves the DNA backbone 5′ to an abasic site, giving a 3′-OH primer for repair synthesis, and mediates other key repair activities. The DNA repair functions are essential for embryogenesis and cell viability. APE1-deficient cells are hypersensitive to DNA-damaging agents, and APE1 is considered an attractive target for inhibitors that could potentially enhance the efficacy of some anti-cancer agents. To enable an important new method for studying the structure, dynamics, catalytic mechanism, and inhibition of APE1, we assigned the chemical shifts (backbone and 13Cβ) of APE1 residues 39-318. We also report a protocol for refolding APE1, which was essential for achieving complete exchange of backbone amide sites for the perdeuterated protein.

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Acknowledgments

This work was supported by a grant from the National Institutes of Health (R01-GM72711 to A.C.D.) and the University of Maryland Marlene and Stewart Greenebaum Cancer Center. B.M. is supported by a Chemistry-Biology Interface (CBI) Training grant from the NIH, T32-GM066706. The NMR spectrometers used in these studies were purchased, in part, with funds from shared instrumentation grants from the NIH (S10-RR10441; S10-RR15741; S10-RR16812; S10-RR23447) and the NSF (DBI 0115795). We thank Mike Morgan for producing the expression plasmid for APE1ΔN38.

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  1. Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland Baltimore, 108 N. Greene St., Baltimore, MD, 21201, USA

    Brittney A. Manvilla, Kristen M. Varney & Alexander C. Drohat

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  1. Brittney A. Manvilla
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  2. Kristen M. Varney
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  3. Alexander C. Drohat
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Correspondence to Alexander C. Drohat.

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Manvilla, B.A., Varney, K.M. & Drohat, A.C. Chemical shift assignments for human apurinic/apyrimidinic endonuclease 1. Biomol NMR Assign 4, 5–8 (2010). https://doi.org/10.1007/s12104-009-9196-y

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  • DOI: https://doi.org/10.1007/s12104-009-9196-y

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