Trinucleotide repeats affect DNA replication in vivo

Abstract

(CGG)n.(CCG)n and (CTG)n.(CAG)n repeats of varying length were cloned into a bacterial plasmid, and the progression of the replication fork through these repeats was followed using electrophoretic analysis of replication intermediates. We observed stalling of the replication fork within repeated DNAs and found that this effect depends on repeat length, repeat orientation relative to the replication origin and the status of protein synthesis in a cell. Interruptions within repeated DNAs, similar to those observed in human genes, abolished the replication blockage. Our results suggest that the formation of unusual DNA structures by trinucleotide repeats in the lagging-strand template may account for the observed replication blockage and have relevance to repeat expansion in humans.

Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells

“…Such intermediates (Fig. 6e) include hairpins 42 or slipped-strand structures [36][37][38] that form between nascent and template strands; between the two nascent strands, which may occur through replication fork reversal [43][44][45] ; or between the two template strands, which may block or pause fork progression 20 . The sequence (CTG or CAG) and the number of repeats at the terminus might affect both the type and the propensity of structure formation.…”

“…Bacterial (Escherichia coli) 19,20 and yeast (Saccharomyces cerevisiae) [21][22][23][24] models support a role for DNA replication in TNR instability in that the direction of replication through the TNR tract affects its stability. In bacterial and yeast systems, repeat deletions predominate regardless of the direction of replication, but the frequency of deletions is higher when the CTG strand, rather than the CAG strand, is the template for lagging-strand synthesis 19 .…”

Long CTG·CAG Repeats from Myotonic Dystrophy Are Preferred Sites for Intermolecular Recombination

“…The expansions and deletions of the TRS due to replication were shown to be dependent on the location of the CTG tracts on the leading (orientation I) or on the lagging (orientation II) strand template (5-7, 33, 42). Also, the orientation of the (CTG⅐CAG) n repeats strongly influenced the pausing of the replication fork in vivo (48).…”

“…Because the two plasmids were introduced successively into the cell, we propose that the replication of the CTG⅐CAG repeats in the pBRW322 derivatives (which were introduced in the first step) influences the recombination frequency. The CTG⅐CAG tracts arrest replication fork progression in vitro and in vivo, presumably due to the formation of unusual secondary structures (48 -50, 67); this occurs predominantly when the CTG tract is located on the lagging strand template (orientation II) (48,77). Hence, we propose a model wherein the stalling of the replication fork at the secondary structures leads to nicks and/or double-strand breaks in the repeating tract.…”

Positioned to expand

“…Formation of these structures by TNRs inhibits the activity of DNA polymerases and other replication proteins in vitro 3 . When the TNRs are longer than the threshold lengths, they impede the DNA replication fork progression in vivo as well, presumably owing to similar structural problems 4 . It seems, therefore, that irregularities of TNR replication could occasionally result in the addition of extra copies of repeats to newly synthesized DNA strands.…”