Fidelity of Primate Cell Repair of a Double-strand Break within a (CTG)·(CAG) Tract

Abstract

At least 15 human diseases are caused by the instability of gene-specific (CTG)⅐(CAG) repeats. The precise mechanism of instability remains unknown, though bacterial and yeast models have suggested a role for aberrant repair of double-strand breaks (DSBs). Using an established primate DSB repair system, we have investigated the fidelity of repair of a DSB within a (CTG)⅐(CAG) repeat tract. DSB repair substrates were generated from plasmids that are stably replicated in their circular form, permitting us to highlight the effects of DSB repair on repeat stability and minimize the contribution of replication. DSBs were introduced into repeat-containing plasmids using a unique BsmI site, such that the entire repeat tract comprised one free end of the linearized plasmid. Substrates containing 17, 47, and 79 repeats, in either their linear duplex form or containing slipped structures (out-of-register interstrand mispairings at repeat sequences), were transiently transfected into primate cells. Linearized plasmids with repeats were repaired with mildly reduced efficiency, while the presence of slipped structures considerably reduced repair efficiency. The repaired products were characterized for alterations within the repeat tract and flanking sequence. DSB repair induced predominantly repeat deletions. Notably, a polarized/ directional deletion effect was observed, in that the repetitive end of the DSB was preferentially removed. This phenomenon was dramatically enhanced when slipped structures were present within the repeat tract, providing the first evidence for error-prone processing of slipped-strand structures. These results suggest the existence of primate nuclease activities that are specific for (CTG)⅐(CAG) repeats and the structures they form.

Hairpin Structure-forming Propensity of the (CCTG·CAGG) Tetranucleotide Repeats Contributes to the Genetic Instability Associated with Myotonic Dystrophy Type 2

“…The products of the sequencing reactions were analyzed on 6% Long Ranger gels (FMC BioProducts) containing 7.5 M urea in the glycerol tolerant gel buffer (1.78 M Tris, 0.57 M taurine, and 0.01 M EDTA) (U. S. Biochemical Corp.). The (CCTG⅐CAGG) 30 is a pure repeat (i.e. it contains no polymorphisms/interruptions) as determined by sequencing of the entire repeat containing tract.…”

“…The ranges of expansions and deletions of the (CCTG⅐CAGG) n repeats are shown; the estimated repeat length varied by Ϯ5 repeats for the plasmids carrying either 200 or 114 repeats. The (CCTG⅐CAGG) 30 , however, was sequenced to indicate the precise number of repeats. In general, the distribution of product sizes within these ranges was random.…”

“…Replication (9, 10, 16 -23), recombination (24 -28), and repair (10,21,29,30) were shown to be responsible for the instabilities of triplet repeat sequences. Slippage of the repeats (31)(32)(33)(34) as promoted by non-B DNA structures (9 -11, 35-37) formed by these repeating sequences causes polymerase to pause during replication, as shown both in vivo as well as in vitro (17, 20, 38 -43), thereby generating instabilities.…”

“…Furthermore, these structures are also recognized by mismatch repair (MMR) (29, 44 -48) and nucleotide excision repair (NER) (49,50); both pathways have been implicated in the stability of the secondary structures, thus influencing the expansion and deletion processes. Also, double strand breaks caused by replication fork arrest or repair of the non-B DNA structures induces repair-mediated recombination that may participate in the expansions observed in both prokaryotic as well as eukaryotic model systems (21,30,(51)(52)(53)(54)(55)(56). Triplet repeat sequences are hotspots for recombination, which may account for the massive expansions found in certain diseases (24 -28, 57, 58).…”

RCPdb: An evolutionary classification and codon usage database for repeat-containing proteins

“…A key feature of trinucleotide repeats (TNRs) is their ability to undergo rapid changes in the number of repeat units during DNA replication and repair (Harding et al 1992;Schlotterer and Tautz 1992;Tachida and Iizuka 1992;Richard et al 2000;Ellegren 2002;Marcadier and Pearson 2003). As TNRs also represent codons, within the coding regions of DNA, homopeptides can thus change in length as a result of TNR length changes.…”