Analysis of gene transcription in cells lacking DNA-PK activity.
The DNA-dependent protein kinase (DNA-PK), comprised of the Ku70/Ku80 (now known as G22p1/Xrcc5) heterodimer and the catalytic subunit DNA-PKcs (now known as Prkdc), is required for the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair. The mechanism of action of DNA-PK remains unclear. We have investigated whether DNA-PK regulates gene transcription in vivo after DNA damage using the subtractive hybridization technique of cDNA representational difference analysis (cDNA RDA). Differential transcription, both radiation-dependent and independent, was detected and confirmed in primary mouse embryo fibroblasts from DNA-PKcs(-/-) and DNA-PKcs(+/+) mice. We present evidence that transcription of the extracellular matrix gene laminin alpha 4 (Lama4) is regulated by DNA-PK in a radiation-independent manner. However, screening of both primary and immortalized DNA-PKcs-deficient cell lines demonstrates that the majority of differences were not consistently dependent on DNA-PK status. Similar results were obtained in experiments using KU mutant hamster cell lines, indicating heterogeneity of transcription between closely related cell lines. Our results suggest that while DNA-PK may be involved in limited gene-specific transcription, it does not play a major role in the transcriptional response to DNA damage.
Bryntesson
F
F
Department of Molecular Haematology and Cancer Biology, Institute of Child Health, University College, London, 30 Guildford Street, London WC1N 1EH, United Kingdom.
Regan
J C
JC
Jeggo
P A
PA
Taccioli
G E
GE
Hubank
M
M
D016475:3T3 Cells; D000818:Animals; D034961:Antigens, Nuclear; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D018076:DNA, Complementary / Q000235:genetics / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D000072200:Ku Autoantigen; D007797:Laminin / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D034961:Antigens, Nuclear; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C501369:LAMA4 protein, human; C501370:Lama4 protein, mouse; D007797:Laminin; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497264:Prkdc protein, mouse; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606512:Xrcc5 protein, mouse; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen