Replication-dependent instability at (CTG) x (CAG) repeat hairpins in human cells

Area of Science:

• Genetics
• Molecular Biology
• Genomics

Background:

• Microsatellite trinucleotide repeat (TNR) instability, particularly (CTG)x(CAG) sequences, underlies numerous neurological and neuromuscular diseases.
• The precise mechanisms driving TNR instability during DNA replication, such as slipped-strand or hairpin formation, remain incompletely understood in human cells.
• Direct in vivo evidence for hairpin structure formation during replication has been lacking.

Purpose of the Study:

• To investigate the formation of hairpin structures during the replication of (CTG)x(CAG) trinucleotide repeats in human cells.
• To provide direct evidence for in vivo hairpin formation during DNA replication.
• To explore factors influencing TNR instability during replication.

Main Methods:

• Creation of isogenic HeLa cell lines using targeted recombination to replicate (CTG)x(CAG) repeats from an ectopic Myc replication origin.
• Analysis of TNR expansion and contraction influenced by origin location and replication strand orientation (leading/lagging).
• Utilizing synthetic zinc finger nucleases for hairpin cleavage to detect hairpin structures in vivo.

Main Results:

• TNR instability was influenced by replication origin location and the leading or lagging strand orientation of the repeats.
• Instability was exacerbated by prolonged cell culture, increased TNR length, and replication inhibition.
• Hairpin cleavage by engineered nucleases provided the first direct evidence of hairpin structure formation during replication in vivo.

Conclusions:

• Hairpin structures are formed during the replication of (CTG)x(CAG) trinucleotide repeats in human cells.
• Replication dynamics, repeat length, and cellular conditions significantly impact TNR instability.
• This study offers critical insights into the molecular mechanisms underlying TNR-associated diseases.