DNA replication and transcription direct a DNA strand bias in the process of targeted gene repair in mammalian cells.
The repair of point mutations can be directed by modified single-stranded DNA oligonucleotides and regulated by cellular activities including homologous recombination, mismatch repair and transcription. Now, we report that DNA replication modulates the gene repair process by influencing the frequency with which either DNA strand is corrected. An SV40-virus-based system was used to investigate the role of DNA synthesis on gene repair in COS-1 cells. We confirm that transcription exerts a strand bias on the gene repair process even when correction takes place on actively replicating templates. We were able to distinguish between the influences of transcription and replication on strand bias by changing the orientation of a gene encoding enhanced green fluorescent protein relative to the origin of replication, and confirmed the previously observed bias towards the untranscribed strand. We report that DNA replication can increase the level of untranscribed strand preference only if that strand also serves as the lagging strand in DNA synthesis. Furthermore, the effect of replication on gene repair frequency and strand bias appears to be independent of certain mismatched base pairs and oligonucleotide length.
Brachman
Erin E
EE
Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA.
Kmiec
Eric B
EB
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001483:Base Sequence; D019556:COS Cells; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D000952:Antigens, Polyomavirus Transforming; D009841:Oligonucleotides; D049452:Green Fluorescent Proteins; D004247:DNA