Mechanisms of fork stalling and reversal at pathogenic CANVAS (AAGGG)n repeats

Replication fork stalling at pathogenic (AAGGG)n repeats in human cells is strictly orientation dependent and linked to G4-DNA formation, with fork reversal driven by SMARCAL1 and RAD51.

• Why it matters: The CANVAS repeat expansion in RFC1 is linked to replication impairment, but the specific fork stalling and reversal mechanisms at the repeat sequence were unclear.
• What they did: Using an SV40-based episome in human cells, 2-dimensional electrophoresis mapped replication intermediates, tested pyridostatin effects, and assessed SMARCAL1 and RAD51 roles; biochemical assays measured SMARCAL1 remodeling with RPA.
• The result: Stalling increased when the homopurine sequence was in the lagging-strand template and with pyridostatin, then reversed forks accumulated; SMARCAL1 remodeled G4-forming repeat forks in ssDNA gaps but not triplex substrates, implying triplex nascent strands at reversed forks.

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