Targeted gene repair activates Chk1 and Chk2 and stalls replication in corrected cells.
Oligonucleotides (ODNs) can direct the exchange of single nucleotides at specific sites in the mammalian genome. It is generally believed that the ODN aligns in homologous register with its complementary site in the target gene and provides a template for the endogenous repair machinery to alter the sequence of the gene. We have been studying the initial phase of the reaction with particular emphasis on the cellular events that occur when the oligonucleotide enters the cell. Our results show that, following introduction of the oligonucleotide, the DNA-damage response pathway is activated, evidenced by the presence of phosphorylated p53, Chk1 and Chk2, respectively. As a result, progression of some of these cells through the cell cycle is slowed and those bearing corrected genes all contain phosphorylated Chk1 and Chk2. In contrast, uncorrected cells contain much lower levels of these proteins in the activated state and pass through the cell cycle in a normal fashion. We suggest that gene repair directed by oligonucleotides activates a pathway that prevents corrected cells from proliferating in cell culture through the activation of Chk1 and Chk2. Our results impact the future use of gene repair for ex vivo gene therapy applications.
Ferrara
Luciana
L
Department of Biological Sciences, University of Delaware, Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711, USA.
Kmiec
Eric B
EB
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics*; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D018274:Electroporation; D004789:Enzyme Activation; D018390:Gene Targeting; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D009841:Oligonucleotides / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016196:S Phase / Q000235:genetics; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D025521:Tumor Suppressor Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases