Induction of V(D)J-mediated recombination of an extrachromosomal substrate following exposure to DNA-damaging agents.
V(D)J recombinase normally mediates recombination signal sequence (RSS) directed rearrangements of variable (V), diversity (D), and joining (J) germline gene segments that lead to the generation of diversified T cell receptor or immunoglobulin proteins in lymphoid cells. Of significant clinical importance is that V(D)J-recombinase-mediated rearrangements at immune RSS and nonimmune cryptic RSS (cRSS) have been implicated in the genomic alterations observed in lymphoid malignancies. There is growing evidence that exposure to DNA-damaging agents can increase the frequency of V(D)J-recombinase-mediated rearrangements in vivo in humans. In this study, we investigated the frequency of V(D)J-recombinase-mediated rearrangements of an extrachromosomal V(D)J plasmid substrate following exposure to alkylating agents and ionizing radiation. We observed significant dose- and time-dependent increases in V(D)J recombination frequency (V(D)J RF) following exposure to ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) but not a nonreactive analogue, methylsulfone (MeSulf). We also observed a dose-dependent increase in V(D)J RF when cells were exposed to gamma radiation. The induction of V(D)J rearrangements following exposure to DNA-damaging agents was not associated with an increase in the expression of RAG 1/2 mRNA compared to unexposed controls or an increase in expression of the DNA repair Ku70, Ku80 or Artemis proteins of the nonhomologous end joining pathway. These studies demonstrate that genotoxic alkylating agents and ionizing radiation can induce V(D)J rearrangements through a cellular response that appears to be independent of differential expression of proteins involved with V(D)J recombination.
Pinsonneault
Robert L
RL
Department of Pediatrics, University of Vermont, Burlington, Vermont, USA.
Vacek
Pamela M
PM
O'Neill
J Patrick
JP
Finette
Barry A
BA
D015415:Biomarkers / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000528:radiation effects; D002460:Cell Line; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005720:Gamma Rays; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D009153:Mutagens / Q000633:toxicity*; D009687:Nuclear Proteins / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic / Q000187:drug effects* / Q000235:genetics* / Q000528:radiation effects; D045567:VDJ Recombinases / Q000378:metabolism*
D015415:Biomarkers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009153:Mutagens; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C064658:RAG-1 protein; D005020:Ethyl Methanesulfonate; D045567:VDJ Recombinases; D045643:DNA Repair Enzymes