Nucleotide excision repair affects the stability of long transcribed (CTG*CAG) tracts in an orientation-dependent manner in Escherichia coli

Several human genetic diseases have been associated with the genetic instability, specifically expansion, of trinucleotide repeat sequences such as (CTG) n ⅐(CAG) n . Molecular models of repeat instability imply replication slippage and the formation of loops and imperfect hairpins in single strands. Subsequently, these loops or hairpins may be recognized and processed by DNA repair systems. To evaluate the potential role of nucleotide excision repair in repeat instability, we measured the rates of repeat deletion in wild type and excision repairdeficient Escherichia coli strains (using a genetic assay for deletions). The rate of triplet repeat deletion decreased in an E. coli strain deficient in the damage recognition protein UvrA. Moreover, loops containing 23 CTG repeats were less efficiently excised from heteroduplex plasmids after their transformation into the uvrA ؊ strain. As a result, an increased proportion of plasmids containing the full-length repeat were recovered after the replication of heteroduplex plasmids containing unrepaired loops. In biochemical experiments, UvrA bound to heteroduplex substrates containing repeat loops of 1, 2, or 17 CAG repeats with a K d of about 10 -20 nM, which is an affinity about 2 orders of magnitude higher than that of UvrA bound to the control substrates containing (CTG) n ⅐(CAG) n in the linear form. These results suggest that UvrA is involved in triplet repeat instability in cells. Specifically, UvrA may bind to loops formed during replication slippage or in slipped strand DNA and initiate DNA repair events that result in repeat deletion. These results imply a more comprehensive role for UvrA, in addition to the recognition of DNA damage, in maintaining the integrity of the genome.

Section: Discussioncontrasting

confidence: 80%

Section: Discussionmentioning

confidence: 95%

Involvement of the Nucleotide Excision Repair Protein UvrA in Instability of CAG·CTG Repeat Sequences in Escherichia coli

Oussatcheva¹,

Hashem²,

Zou³

et al. 2001

“…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).…”

mentioning

confidence: 99%

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

Dere

Напиерала

Ranum

et al. 2004

The genetic instabilities of (CCTG⅐CAGG) n tetranucleotide repeats were investigated to evaluate the molecular mechanisms responsible for the massive expansions found in myotonic dystrophy type 2 (DM2) patients. DM2 is caused by an expansion of the repeat from the normal allele of 26 to as many as 11,000 repeats. Genetic expansions and deletions were monitored in an African green monkey kidney cell culture system (COS-7 cells) as a function of the length (30, 114, or 200 repeats), orientation, or proximity of the repeat tracts to the origin (SV40) of replication. As found for CTG⅐CAG repeats related to DM1, the instabilities were greater for the longer tetranucleotide repeat tracts. Also, the expansions and deletions predominated when cloned in orientation II (CAGG on the leading strand template) rather than I and when cloned proximal rather than distal to the replication origin. Biochemical studies on synthetic d(CAGG) 26 and d(CCTG) 26 as models of unpaired regions of the replication fork revealed that d(CAGG) 26 has a marked propensity to adopt a defined base paired hairpin structure, whereas the complementary d(CCTG) 26 lacks this capacity. The effect of orientation described above differs from all previous results with three triplet repeat sequences (including CTG⅐CAG), which are also involved in the etiologies of other hereditary neurological diseases. However, similar to the triplet repeat sequences, the ability of one of the two strands to form a more stable folded structure, in our case the CAGG strand, explains this unorthodox "reversed" behavior.

“…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.…”

mentioning

confidence: 99%

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

Pluciennik

Iyer

Напиерала

et al. 2002

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...