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First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01241

Journal of Cell Science 117, 3579-3592 (2004)
Published by The Company of Biologists 2004
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Research Article

CPD-photolyase adenovirus-mediated gene transfer in normal and DNA-repair-deficient human cells

Vanessa Chiganças1, Alain Sarasin2 and Carlos Frederico Martins Menck1,*

1 Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1374. Ed. Biomédicas 2, São Paulo, 05508-900, Brazil
2 Laboratory of Genetic Instability and Cancer, UPR 2169, Institut Gustave-Roussy, 94805 Villejuif CEDEX 05, France

* Author for correspondence (e-mail: cfmmenck{at}usp.br)

Accepted 30 March 2004

Cyclobutane pyrimidine dimers (CPDs) are the most frequent and deleterious lesions generated in the mammalian genome after UV-C irradiation. The persistence of these lesions in DNA can be toxic and mutagenic, and also represents a specific signal to apoptosis. To investigate the CPDs repair in situ and consequent UV-induced apoptosis in human cells, we generated a recombinant adenovirus vector containing the gene encoding a CPD-photolyase-EGFP fusion protein (Adphr-EGFP). Adphr-EGFP-infected cells are proficient in photorepair, which prevents apoptotic cell death in comparison with samples kept in the dark, indicating that the fusion protein is functional in CPD recognition and removal. By using local UV irradiation, foci of the photolyase fusion protein were observed in UV-damaged areas of the nuclei in colocalization with NER enzymes. Phr-EGFP migration to CPD sites and redistribution after photorepair was followed, and shown to present similar kinetics in normal or DNA-repair-deficient cells. To our knowledge, this is the first report of an investigation of CPDs repair in situ employing a CPD-photolyase-EGFP enzyme. The Adphr-EGFP vector can be an informative tool to investigate the repair and cellular consequences of UV-induced lesions in primary human cells.

Key words: Cyclobutane pyrimidine dimers, Adenovirus, Photorepair, Local UV irradiation, Apoptosis




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[Abstract] [PDF]


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