CPD-photolyase adenovirus-mediated gene transfer in normal and DNA-repair-deficient human cells.
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.
Chiganças
Vanessa
V
Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1374. Ed. Biomédicas 2, São Paulo 05508-900, Brazil.
Sarasin
Alain
A
Menck
Carlos Frederico Martins
CF
D000256:Adenoviridae / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004260:DNA Repair; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008190:Lyases / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014466:Ultraviolet Rays
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008190:Lyases