Gene repair in mammalian cells is stimulated by the elongation of S phase and transient stalling of replication forks.
The repair of point mutations directed by modified single-stranded DNA oligonucleotides is dependent on the activity of proteins involved in homologous recombination (HR). As a consequence, factors that stimulate homologous recombination, such as double strand breaks, can impact the frequency with which repair occurs. Here, we report that the stalling of replication forks can also activate the gene repair pathway and lead to an enhanced level of nucleotide exchange. The mammalian cell line, DLD-1, containing an integrated mutant eGFP gene, was used as an assay system to explore how replication fork activity affects the overall repair reaction. The addition of 2',3'-dideoxycytidine (ddC), a nucleoside analog that retards the rate of elongation and effectively stalls the replication fork, results in a lengthened S phase and an increased number of gene repair events. This stimulation was reversed when caffeine was added to the reaction at concentrations that block the homologous recombination pathway. In contrast, the nucleoside analog, 1-beta-D-arabinofuranosylcytosine which stops replication in these cells, failed to stimulate the gene repair reaction to any appreciable degree until the block is released and active replication resumes. Furthermore, overexpression of wild-type p53 which is known to bind transiently to stalled replication forks blocked the stimulatory effect of ddC. Overexpression of mutant p53 genes, deficient in the capacity to bind DNA, however, did not inhibit the reaction. Our results indicate that an expansion of S phase and a transient stalling of replication forks can increase the frequency of targeted gene repair.
Brachman
Erin E
EE
Department of Biological Sciences, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19716, USA.
Kmiec
Eric B
EB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D016049:Didanosine / Q000494:pharmacology; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D010441:Peptide Chain Elongation, Translational; D017354:Point Mutation; D016196:S Phase / Q000187:drug effects; D016047:Zalcitabine / Q000494:pharmacology
D049452:Green Fluorescent Proteins; D016047:Zalcitabine; D016049:Didanosine