Expansion and Deletion of Triplet Repeat Sequences inEscherichia coli Occur on the Leading Strand of DNA Replication

Abstract

Expansions and deletions of triplet repeat sequences that cause human hereditary neurological diseases were previously suggested to be mediated by the formation of DNA hairpins on the lagging strand during replication. The replication properties of CTG⅐CAG, CGG⅐CCG, and TTC⅐GAA repeats were studied in Escherichia coli using an in vivo phagemid system as a model for continuous leading strand synthesis. The repeats were substantially deleted when the CTG, CGG, and GAA repeats were the templates for rolling circle replication from the f1 phage origin. The deletions may be mediated by hairpins formed by these repeat tracts. The distributions of the deletion products of the CTG⅐CAG and CGG⅐CCG tracts indicated that hairpins of discrete sizes mediate deletions during complementary strand synthesis. Deletions during rolling circle synthesis are caused by larger hairpins of specific sizes. Thus, most deletion products were of defined lengths, suggesting a preference for specific hairpin intermediates. Small expansions of the CTG⅐CAG and CGG⅐CCG repeats were also observed, presumably due to the formation of CTG and CGG hairpins on the nascent complementary strand. Since rolling circle replication has been established in vitro as a model for leading strand synthesis, we conclude that triplet repeat instability can also occur on the leading strand of DNA replication.The genetic instability of three triplet repeat sequences (TRS), 1 CTG⅐CAG, CGG⅐CCG, and TTC⅐GAA, has been shown to result in approximately 12 hereditary neurological diseases (1-3) including myotonic dystrophy (4), Kennedy's disease (5), fragile X disease (6), and Freidreich's ataxia (7). These diseases are inherited in a non-Mendelian fashion by a phenomenon called "anticipation," which is characterized by an increase in severity and a decrease in age of onset from one generation to the next. The anomalous expansion of TRS has been identified as the molecular basis for anticipation (1). Triplet repeat tracts are highly polymorphic and have been shown to range from five to approximately 40 repeats in normal human chromosomes. Expansion of these tracts can result in type I and type II diseases as classified by Paulson and Fischbeck (3). Whereas type I diseases are characterized by modestly expanded TRS (approximately 30 -80 repeats) in the coding region of a gene, type II diseases contain massive expansions (Ͼ1000 repeats) of the triplet repeat tract in the 5Ј-UTR or 3Ј-UTR or in an intron of a gene (1, 3).Escherichia coli has been used as a genetically tractable system for the study of TRS in vivo (8). The genetic instability of TRS in E. coli is dependent on its orientation relative to the unidirectional ColE1 origin of replication as well as host cell growth phase and transcription (9 -13). Other factors including genetic background (12), methyl-directed mismatch repair (14), and expression of single-stranded DNA binding protein (SSB) (15) are also important. The effect of the orientation of the TRS with respect to the direction of replication on its instabi...

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

“…Furthermore, no rearrangements were observed on analyzing individual colonies obtained from transforming the parental plasmids into E. coli HB101. Likewise, rearrangements were not observed previously in other prior instability studies with triplet repeat inserts in E. coli HB101 (10,16,18,19,39,86,(91)(92)(93). This confirms that the rearrangements were because of an intrinsic property of the pcDNA3.1 shuttle vector that carries the SV40 origin of replication.…”

“…In case of the tetranucleotide repeats, however, instability refers to both expansions and deletions. Thus, upon initial consideration, our results appear to be similar to the results with the triplet repeat sequences, including CTG⅐CAG, CGG⅐CCG, and GAA⅐TTC, in which orientation II was shown to be more unstable (9,10,16,18,19,21,22,39,52,85,86,95,96). However, the CCTG sequences are genetically unstable in the orientation prone to expand (orientation II) (Fig.…”

“…Previous studies showed a marked effect of orientation of trinucleotide repeats (CTG⅐CAG, CGG⅐CCG, and GAA⅐TTC) with respect to the origin of replication (10,16,18,19,21,39,85,86). Therefore, we studied plasmids carrying the (CCTG⅐CAGG) repeats in both orientations.…”

The Myotonic Dystrophy Type 1 Triplet Repeat Sequence Induces Gross Deletions and Inversions

“…The highest frequency was found in JJC510 (14,433 white CFUs out of 25,707 total CFUs) (0.561). A somewhat lower fraction was found in KMBL1001 cells (8,152 white CFUs out of 22,340 total viable cells) (0.365) (Table III). A similar fraction of deleted mutants was detected in strains RW118 and AB1157 (2,961 white CFUs out of a total of 15,065 CFUs (0.196) and 4,165 out of 19,014 (0.219), respectively).…”

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

“…DNA replication (5)(6)(7)(8) and repair including methyl-directed mismatch repair (9 -11), nucleotide excision repair (12), DNA polymerase III exonucleolytic proofreading (13), and double-strand break repair (14) have been implicated.…”