Modulation of transcription reveals a new mechanism of triplet repeat instability in Escherichia coli.

Many human hereditary disease genes are associated with the expansion of triplet repeat sequences. In Escherichia coli (CTG/CAG) triplet repeat sequences are unstable and we have developed a plasmid-based assay enabling us to observe and quantify both expansions and deletions. In this work, we have investigated the role of transcription on the instability of a (CTG/CAG) insert containing 64 repeats. Using this assay, we show that induction of transcription results in a significant increase in the frequency of long deletions and a reduction in the frequency of long expansions. On the other hand, overproduction of transcription repressor molecules leads to an increase in both expansions and deletions. In this latter case, we propose that the increased instability is due to the arrest of replication progression by the interaction of the repressor molecule with its cognate operator and subsequent generations of DNA strand breaks.

Mechanisms of dinucleotide repeat instability in Escherichia coli.

The high level of polymorphism of microsatellites has been used for a variety of purposes such as positional cloning of genes associated with diseases, forensic medicine, and phylogenetic studies. The discovery that microsatellites are associated with human diseases, not only as markers of risk but also directly in disease pathogenesis, has triggered a renewed interest in understanding the mechanism of their instability. In this work we have investigated the role of DNA replication, long patch mismatch repair, and transcription on the genetic instability of all possible combinations of dinucleotide repeats in Escherichia coli. We show that the (GpC) and (ApT) self-complementary sequence repeats are the most unstable and that the mode of replication plays an important role in their instability. We also found that long patch mismatch repair is involved in avoiding both short deletion and expansion events and also in instabilities resulting from the processing of bulges of 6 to 8 bp for the (GpT/ApC)- and (ApG/CpT)- containing repeats. For each dinucleotide sequence repeat, we propose models for instability that involve the possible participation of unusual secondary structures.