Replication-dependent instability at (CTG) x (CAG) repeat hairpins in human cells.
Instability of (CTG) x (CAG) microsatellite trinucleotide repeat (TNR) sequences is responsible for more than a dozen neurological or neuromuscular diseases. TNR instability during DNA synthesis is thought to involve slipped-strand or hairpin structures in template or nascent DNA strands, although direct evidence for hairpin formation in human cells is lacking. We have used targeted recombination to create a series of isogenic HeLa cell lines in which (CTG) x (CAG) repeats are replicated from an ectopic copy of the Myc (also known as c-myc) replication origin. In this system, the tendency of chromosomal (CTG) x (CAG) tracts to expand or contract was affected by origin location and the leading or lagging strand replication orientation of the repeats, and instability was enhanced by prolonged cell culture, increased TNR length and replication inhibition. Hairpin cleavage by synthetic zinc finger nucleases in these cells has provided the first direct evidence for the formation of hairpin structures during replication in vivo.
Liu
Guoqi
G
Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA.
Chen
Xiaomi
X
Bissler
John J
JJ
Sinden
Richard R
RR
Leffak
Michael
M
D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004261:DNA Replication / Q000502:physiology*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D053842:Microsatellite Instability; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics / Q000502:physiology; D018911:Trinucleotide Repeats / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology
D016271:Proto-Oncogene Proteins c-myc; D004247:DNA; D004720:Endonucleases