Nucleotide excision repair proteins and their importance for radiation-enhanced transfection.
OBJECTIVE
Irradiated cells transfect more efficiently than unirradiated cells because of a radiation-induced increase in plasmid integration. However, the molecular mechanism is unclear. Because of recent observations that nucleotide excision repair (NER) proteins can be involved in certain types of recombination in yeast, it was hypothesized that NER proteins might play a role in this radiation-enhanced integration.


METHODS
Hamster and human cells with inactivating mutations in NER genes were irradiated at doses from 0 to 6 Gy and then immediately transfected with a linearized selectable marker plasmid. Transfection-enhancement ratios (TERs) were calculated as the ratio of the number of drug-resistant colonies in unirradiated cells to the number of transfectants in irradiated cells, corrected for cytotoxicity from radiation.


RESULTS
Transfection into unirradiated rodent cells was unaffected by NER mutation status. Transfection into unirradiated human cells, however, was increased by NER mutation. The TERs were 5 and 100 for CHO and primary human fibroblasts, respectively, after exposure of the cells to 6 Gy. Mutations in ERCC1, XPA, XPB, XPC, XPF, XPG and CSB dramatically reduced TER. Mutations in ERCC1, XPC, XPF, XPG and CSB suppressed transfection so that the TER was significantly below 1.


CONCLUSIONS
The mechanism of radiation-enhanced plasmid integration was distinct from that of plasmid integration in unirradiated cells, and NER gene products were critical for enhanced integration to occur.
Nimura
Y
Y
Department of Experimental Radiation Oncology, University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA.
Prithivarajsingh
S
S
Ismail
S M
SM
Nairn
R S
RS
Tucker
S L
SL
Allen
P K
PK
Stevens
C W
CW
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000502:physiology; D029721:Drosophila Proteins / Q000502:physiology; D004720:Endonucleases; D006801:Humans; D009154:Mutation; D010957:Plasmids; D011506:Proteins / Q000502:physiology; D011995:Recombination, Genetic / Q000528:radiation effects*; D014162:Transfection
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D011506:Proteins; C095888:xeroderma pigmentosum group F protein; C078672:hay protein, Drosophila; C071447:ERCC1 protein, human; D004720:Endonucleases