A role for the human DNA repair enzyme HAP1 in cellular protection against DNA damaging agents and hypoxic stress (original) (raw)
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Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital
Oxford 0X3 9DU, UK
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Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital
Oxford 0X3 9DU, UK
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,
Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital
Oxford 0X3 9DU, UK
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Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital
Oxford 0X3 9DU, UK
* To whom correspondence should be addressed
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Received:
21 September 1994
Revision received:
19 October 1994
Accepted:
19 October 1994
Published:
25 November 1994
Cite
Lisa J. Walker, Randa B. Craig, Adrian L. Harris, Ian D. Hickson, A role for the human DNA repair enzyme HAP1 in cellular protection against DNA damaging agents and hypoxic stress, Nucleic Acids Research, Volume 22, Issue 23, 25 November 1994, Pages 4884–4889, https://doi.org/10.1093/nar/22.23.4884
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Abstract
The HAP1 protein (also known as APE/Ref-1) is a bifunctional human nuclear enzyme required for repair of apurinic/apyrimidinic sites in DNA and reactivation of oxidized proto-oncogene products. To gain insight into the biological roles of HAP1, the effect of expressing antisense HAP1 RNA in HeLa cells was determined. The constructs for antisense RNA expression consisted of either a full-length HAP1 cDNA or a genomic DNA fragment cloned downstream of the CMV promoter in pcDNAneo. Stable HeLa cell transfectants expressing HAP1 antisense RNA were found to express greatly reduced levels of the HAP1 protein compared to equivalent sense orientation and vector-only control transfectants. The antisense HAP1 transfectants exhibited a normal growth rate, cell morphology and plating efficiency, but were hypersensitive to killing by a wide range of DNA damaging agents, including methyl methanesulphonate, hydrogen peroxide, menadione, and paraquat. However, survival after UV irradiation was unchanged. The antisense transfectants were strikingly sensitive to changes in oxygen tension, exhibiting increased killing compared to controls following exposure to both hypoxia (1 % oxygen) and hyperoxia (100% oxygen). Consistent with a requirement for HAP1 in protection against hypoxic stress, expression of the HAP1 protein was found to be induced in a time-dependent manner in human cells during growth under 1% oxygen. The possible involvement of a depletion of cellular glutathione being linked to the hypoxic stress-sensitive phenotype of the antisense HAP1 transfectants came from the finding that they also exhibited hypersensitivity to buthionine sulphoximine, an inhibitor of glutathione biosynthesis. We conclude that the HAP1 protein is a key factor in cellular protection against a wide variety of cellular stresses, including DNA damage and a change in oxygen tension.
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