Quantifying Replication Fork Progression at CTG Repeats by 2D Gel Electrophoresis.

Abstract

Physical separation of branched DNA from linear molecules is based on the difference of mobility of linear versus branched DNA during two-dimensional agarose gel electrophoresis. Structured DNA migrates as slower species when compared to linear DNA of similar molecular weight. Metabolic processes such as S phase replication or double strand-break repair may generate branched DNA molecules. Trinucleotide repeats are naturally prone to form secondary structures that can modify their migration through an agarose gel matrix. These structures may also interfere in vivo with replication, by slowing down replication-fork progression, transiently stalling forks, possibly leading to secondary structure such as Holliday junctions or hemicatenanes. Alternatively, reversed replication forks may occur following fork stalling, disrupting replication dynamics and modifying DNA migration on agarose gel. So although two-dimensional agarose gel electrophoresis theoretically allows to resolve a mixture of structured DNA molecules and quantify them by radioactive hybridization, its practical application to trinucleotide repeats faces some serious technical challenges.