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

Abstract

Homologous recombination was shown to enable the expansion of CTG⅐CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG⅐CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG⅐CAG) n repeats apparently recombine with an ϳ60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG⅐CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG⅐CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.Micro-and minisatellite instability has been associated with human genetic diseases (1-4). Approximately 14 hereditary neurological diseases are caused by the genetic instabilities of triplet repeat sequences (TRS) 1 in or near relevant genes (reviewed in Ref. 1). Long tracts of repeating CTG⅐CAG sequences are responsible for myotonic dystrophy and several other diseases. Normal individuals have 5-37 repeats in the myotonic dystrophy protein kinase gene, whereas the mutations (expansions) can be in the range of 50 -3000 repeats. Thus, an explosive allele length change during intergenerational transmission can occur in this autosomal dominant disease, thus causing an increase in the severity of the disease and a decrease of the age at onset (anticipation).The mechanisms of genetic instabilities have been widely investigated in the past 6 years (1). DNA replication (5-8) 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.Repetitive sequences promote homologous recombination in prokaryotic as well as eukaryotic systems, presumably by virtue of forming unusual DNA secondary structures (15)(16)(17)(18)(19)(20)(21)(22). In fact, homologous recombination has been implicated in the instability of repetitive sequences (23-26). Similar findings have also been made with CTG⅐CAG repeats using a two-plasmid system in Escherichia coli (27,28). Multiple fold expansions, deletions, and the exchange of point mutations between tracts were found in this system; these events were dependent on the presence of TRS tracts on both plasmids, CTG⅐CAG repeat lengths longer than 30, and a functional recA gene.Previously (29), it was proposed that CTG⅐CAG tracts might function as recombination hot spots in the bovine genome. More recently, Young et al. (30) surveyed the genome of Saccharomyces cerevisiae and suggested that these repeats cou...

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

“…Oligonucleotide Model Studies: Enzymatic Probing-Two oligonucleotides, d(CAGG) 26 and d(CCTG) 26 , were chemically synthesized to study their structural properties as related to the behavior of unpaired regions of the (CCTG⅐CAGG) repeats during replication and related processes that unwind the duplex. d(CAGG) 26 and d(CCTG) 26 were purified and labeled ("Experimental Procedures").…”

“…The individual oligonucleotides (Genosys), d(CCTG) 26 and d(CAGG) 26 , were purified on a 6% denaturing polyacrylamide gel containing 7.5 M urea in glycerol tolerant gel buffer (U. S. Biochemical Corp.). The purified oligonucleotides were labeled at the 5Ј end with 15 units of T4 polynucleotide kinase (U. S. Biochemical Corp.) and [␥-32 P]ATP at 37°C for 1 h. The labeled oligonucleotides were purified on a 6% denaturing polyacrylamide gel.…”

“…Chemical Modifications and Enzymatic Probing-Three chemical probes, osmium tetraoxide (OsO 4 ) (Aldrich), potassium permanganate (KMnO 4 ) (Fisher), and diethyl pyrocarbonate (DEPC) (Sigma), each were used to modify the d(CCTG) 26 oligonucleotide, whereas the latter two chemicals were used to modify the d(CAGG) 26 oligonucleotide. The purified and labeled oligonucleotides (4 -5 ϫ 10 5 cpm/reaction) in 10 mM Tris, 40 mM NaCl, and 10 mM MgCl 2 were denatured by heating at 80°C for 5 min followed by renaturation by gradually decreasing the temperature (2°C/min) to the indicated reaction temperature (61).…”

Binding Modes of the Initiator and Inhibitor Forms of the Replication Protein π to the γ ori Iteron of Plasmid R6K

“…Strains and Plasmids-Escherichia coli strain ECF001 contains a chromosomal copy of the pir gene under an arabinose-inducible promoter, Para BAD (22). Construction of plasmids pFW25 (23) and pRK1 (20) was previously described.…”

“…5. To conduct our experiments, we used an E. coli strain (ECF001) that carried an arabinose-inducible pir gene on its chromosome (22); it also harbored the ␥ ori containing plasmid, pFW25. In this strain, the replication rate of plasmid pFW25 rises with increasing levels of supplied arabinose (and then levels off).…”