Restarted replication forks are error-prone and cause CAG repeat expansions and contractions

Area of Science:

• Molecular Biology
• Genetics
• DNA Replication

Background:

• Trinucleotide repeats form secondary DNA structures that impede DNA replication and repair.
• Replication stress and fork stalling are implicated in repeat expansions and contractions, but the precise mechanisms remain unclear.

Purpose of the Study:

• To investigate the fidelity of restarted replication forks through trinucleotide repeat tracts.
• To determine the contribution of recombination-dependent replication (RDR) to repeat instability after fork stalling.

Main Methods:

• Utilized a site-specific replication fork barrier (RFB) system in fission yeast.
• Introduced long CAG repeat tracts at varying distances and orientations relative to the RFB.
• Assessed repeat instability using molecular assays to measure expansions and contractions.

Main Results:

• Replication by restarted forks showed low fidelity through CAG repeat tracts located 2-7 kb from the RFB.
• Elevated, Rad52- and Rad8ScRad5/HsHLTF-dependent, repeat instability (expansions and contractions) was observed.
• Instability was less pronounced when repeat tracts were immediately before or after the barrier, suggesting the restarted fork's traversal is key.

Conclusions:

• Replication restart via RDR is a major source of trinucleotide repeat instability, specifically through long repeat tracts.
• The fidelity of the restarted fork, rather than fork reversal or initial stall events, is the primary driver of repeat instability.
• Replication stress tolerance through fork restart comes at the expense of increased repetitive sequence instability.