Adenovirus mediated transduction of the human DNA polymerase eta cDNA.
Xeroderma pigmentosum (XP) is an autosomal recessive photosensitive disorder with an extremely high incidence of skin cancers. Seven complementation groups, corresponding to seven proteins involved in nucleotide excision repair (NER), are associated with this syndrome. However, in XP variant patients, the disorder is caused by defects in DNA polymerase eta; this error prone polymerase, encoded by POLH, is involved in translesion DNA synthesis (TLS) on DNA templates damaged by ultraviolet light (UV). We constructed a recombinant adenovirus carrying the human POLH cDNA linked to the EGFP reporter gene (AdXPV-EGFP) and infected skin fibroblasts from both XPV and XPA patients. Twenty-four hours after infection, the DNA polymerase eta-EGFP fusion protein was detected by Western blot analysis, demonstrating successful transduction by the adenoviral vector. Protein expression was accompanied by reduction in the high sensitivity of XPV cells to UV, as determined by cell survival and apoptosis-induction assays. Moreover, the pronounced UV-induced inhibition of DNA synthesis in XPV cells and their arrest in S phase were attenuated in AdXPV-EGFP infected cells, confirming that the transduced polymerase was functional. However, over-expression of polymerase eta mediated by AdXPV-EGFP infection did not result in enhancement of cell survival, prevention of apoptosis, or higher rate of nascent DNA strand growth in irradiated XPA cells. These results suggest that TLS by DNA polymerase eta is not a limiting factor for recovery from cellular responses induced by UV in excision-repair deficient fibroblasts.
Lima-Bessa
Keronninn Moreno
KM
Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1374 São Paulo, SP 05508-900, Brazil.
Chiganças
Vanessa
V
Stary
Anne
A
Kannouche
Patricia
P
Sarasin
Alain
A
Armelini
Melissa Gava
MG
de Fátima Jacysyn
Jacqueline
J
Amarante-Mendes
Gustavo P
GP
Cordeiro-Stone
Marila
M
Cleaver
James E
JE
Menck
Carlos Frederico Martins
CF
D000256:Adenoviridae / Q000235:genetics*; D015153:Blotting, Western; D002478:Cells, Cultured; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics* / Q000528:radiation effects; D018076:DNA, Complementary / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D014161:Transduction, Genetic / Q000379:methods*; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*; D000099270:Y-Family DNA Polymerases
D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase; D049452:Green Fluorescent Proteins; C485184:enhanced green fluorescent protein; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases