The rate of extrachromosomal homologous recombination within a novel reporter plasmid is elevated in cells lacking functional ATM protein.
Homologous recombination between identical stretches of DNA depends on the coordinated action of many tightly regulated proteins. Cellular defects in homologous recombination are strongly associated with increased genomic instability and tumorigenesis. In cells of the cancer-prone syndrome ataxia telangiectasia (A-T), increased intrachromosomal recombination has been demonstrated, while extrachromosomal recombination has been discussed controversially. We constructed a novel, episomally replicating pGrec recombination vector containing two mutated alleles of the enhanced green fluorescent protein (eGFP) gene. Homologous recombination can reconstitute functional wildtype eGFP, thus allowing detection of recombination events based on cellular eGFP fluorescence. Using an isogenic cell pair of A-T fibroblasts and derivatives complemented by an ATM expression vector, we were able to demonstrate in A-T cells high extrachromosomal recombination rates, which are suppressed upon ectopic ATM expression. We thus found that ATM deficiency increases spontaneous recombination not only in intrachromosomal but also in extrachromosomal substrates, suggesting that lack of ATM increases homologous recombination independent of the chromatin structure.
Drexler
Guido A
GA
Institute of Molecular Radiobiology, D-85758 Neuherberg, Germany.
Wilde
Susanne
S
Beisker
Wolfgang
W
Ellwart
Joachim
J
Eckardt-Schupp
Friederike
F
Fritz
Eberhard
E
D000818:Animals; D001260:Ataxia Telangiectasia / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D016466:CHO Cells; D018797:Cell Cycle Proteins; D002875:Chromosomes / Q000235:genetics; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000172:deficiency*; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000502:physiology*; D010957:Plasmids / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency*; D011995:Recombination, Genetic; D025521:Tumor Suppressor Proteins / Q000172:deficiency*
D018797:Cell Cycle Proteins; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases