Academic literature on the topic 'Trinucleotide repeat instability'

In order to identify cellular factors that affect trinucleotide repeat stability, changes in the length of a (CTG)₄₃ repeat were studied over 140 generations in wild-type <i>Escherichia coli</i> and in strains that are deficient in post-replicative mismatch repair, secondary structure repair and homologous recombination. It is shown that (CTG)₄₃ inserted into pUC18 expands and contracts in wild-type <i>E. coli</i> in an orientation-dependent manner that is unaffected by transcription. In cells deficient in post-replicative mismatch repair (CTG)₄₃ repeat instability is greater than in wild-type cells but orientation-independent. The observation of single trinucleotide insertions and deletions in these mutator mutants indicates that replication slippages of 3 bp occur <i>in vivo</i> leading to repeat expansion and contraction if left unrepaired. Compared to wild-type cells large deletions are reduced in these mutator mutants, but only if the CTG sequence serves as the lagging strand. Based on the opposing effects of mismatch repair a model is proposed in which orientation-dependent CTG repeat instability in mismatch repair proficient cells is caused by the repair of 3-bp slippages. This leads to the creation of larger deletions during repair synthesis due to the formation of unusual secondary structures by the CTG sequence on the lagging strand. Mutations in the <i>recA</i> and <i>sbcCD </i>genes do not affect the stability of plasmid-borne CTG repeats. Similarly the viability of <i>recA</i>-deficient strains carrying chromosomal insertions of (CTG)₂₅ and (CTG)₄₃ suggests that, unlike long palindromes, these trinucleotide repeats are not substrates for the structure-directed nuclease complex SbcCD or, alternatively, they do not form secondary structures frequently enough to cause lethality in <i>recA-</i>deficient hosts. In contrast, a mutation in the <i>recG</i> gene, also involved in homologous recombination, severely destabilises the (CTG)₄₃ repeat in a strongly orientation-dependent manner that exceeds all other tested mutants. Possible explanations for this observation are discussed.

In order to identify the molecular basis of genetic instability, a polymerization-independent strategy is developed to generate expanded repeat arrays. The repeat tracts are integrated in the 5’end of <i>lacZ</i> gene in the <i>Escherichia coli</i> chromosome. Using this model system, instability is studied in wild type <i>E. coli</i> and in strains deficient in cellular pathways such as DNA repair, replication and recombination. The work demonstrates that instability (expansion and contraction) in wild type cells is length and orientation dependent. Longer tracts are more unstable than shorter ones and the orientation where CAG repeats are on the leading strand template is more unstable than the opposite where CTG repeats are on the leading strand template. This orientation-dependence of CAG·CTG trinucleotide repeat instability is determined by the proofreading subunit of DNA polymerase II (DnaQ) in the presence of the hairpin nuclease SbcCD. The analysis of the sizes of deletions observed in wild type and mutant cells is consistent with the formation of secondary structures <i>in vivo</i>. The mismatch repair pathway does not affect the instability of CTG repeats in the <i>E. coli</i> chromosome but influences the CAG orientation. It is suggested that MutS stabilizes CAG repeats by initiating a “repair” process and protecting hairpins from SbcCD, which can cleave hairpins in the presence of MutL and MutH. Finally, the roles of two helicases, Rep and UvrD are analyzed. A mutation in <i>rep</i> helicase strongly destabilizes CTG repeats with no effect on the CAG orientation UvrD mutants show instability in both orientations. The increase in instability in the <i>uvrD</i> mutant depends on RecF in the CTG orientation.

Genomic instability, in the form of gene mutations, insertions/deletions, and gene amplifications, is one of the hallmarks in many types of cancers and other inheritable genetic disorders. Trinucleotide repeat (TNR) disorders, such as Huntington’s disease (HD) and Myotonic dystrophy (DM) can be inherited and repeats may be extended through subsequent generations. However, it is not clear how the CAG repeats expand through generations in HD. Two possible repeat expansion mechanisms include: 1) polymerase mediated repeat extension; 2) persistent TNR hairpin structure formation persisting in the genome resulting in expansion after subsequent cell division. Recent in vitro studies suggested that a family A translesion polymerase, polymerase θ (Polθ), was able to synthesize DNA larger than the template DNA. Clinical and in vivo studies showed either overexpression or knock down of Polθ caused poor survival in breast cancer patients and genomic instability. However, the role of Polθ in TNR expansion remains unelucidated. Therefore, we hypothesize that Polθ can directly cause TNR expansion during DNA synthesis. The investigation of the functional properties of Polθ during DNA replication and TNR synthesis will provide insight for the mechanism of TNR expansion through generations.

Trinucleotide repeat (TNR) instability including repeat expansions and repeat deletions is the cause of more than 40 inherited incurable neurodegenerative diseases and cancer. TNR instability is associated with DNA damage and base excision repair (BER). In this dissertation research, we explored the mechanisms of BER-mediated TNR instability via biochemical analysis of the BER protein activities, DNA structures, protein-protein interaction, and protein-DNA interaction by reconstructing BER in vitro using synthesized oligonucleotide TNR substrates and purified human proteins. First, we evaluated a germline DNA polymerase β (pol β) polymorphic variant, pol βR137Q, in leading TNR instability-mediated cancers or neurodegenerative diseases. We find that the pol βR137Q has slightly weaker DNA synthesis activity compared to that of wild-type (WT) pol β. Because of the similar abilities between pol βR137Q and WT pol β in bypassing a template loop structure, both pol βR137Q and WT pol β induces similar amount of repeat deletion. We conclude that the slightly weaker DNA synthesis activity of pol βR137Q does not alter the TNR instability compared to that of WT pol β, suggesting that the pol βR137Q carriers do not have an altered risk in developing TNR instability-mediated human diseases. We then investigated the role of DNA synthesis activities of DNA polymerases in modulating TNR instability. We find that pol βY265C and pol ν with very weak DNA synthesis activities predominantly promote TNR deletions. We identify that the sequences of TNRs may also affect DNA synthesis and alter the outcomes of TNR instability. By inhibiting the DNA synthesis activity of pol β using a pol β inhibitor, we find that the outcome of TNR instability is shifted toward repeat deletions. The results provide the direct evidence that DNA synthesis activity of DNA polymerases can be utilized as a potential therapeutic target for treating TNR expansion diseases. Finally, we explored the role of post-translational modification (PTM) of proliferating cell nuclear antigen (PCNA) on TNR instability. We find that ubiquitinated PCNA (ub-PCNA) stimulates Fanconi associated nuclease 1 (FAN1) 5’-3’ exonucleolytic activities directly on hairpin structures, coordinating flap endonuclease 1 (FEN1) in removing difficult secondary structures, thereby suppressing TNR expansions. The results suggest a role of mono-ubiquitination of PCNA in maintaining TNR stability by regulating nucleases switching. Our results suggest enzymatic activities of DNA polymerases and nucleases and the regulation of the activities by PTM play important roles in BER-mediated TNR instability. This research provides the molecular basis for future development of new therapeutic strategies for prevention and treatment of TNR-mediated neurodegenerative diseases.

The expansion of trinucleotide repeat tracts is the cause of nearly twenty genetic disorders. Almost all these diseases are characterised by anticipation, which means an earlier age of onset and an increased severity of the symptoms from one generation to the next. The mechanisms of trinucleotide repeat expansion are not understood. In the course of this project, I have investigated the instability of a trinucleotide repeat array of 43 copies integrated at the <i>attB </i>site of chromosomes of various <i>Escherichia coli </i>mutants. The trinucleotide repeat tract (CTG)₄₃was integrated into the <i>E. coli </i>chromosome in both possible orientations using an intermediate vector and exploiting site-specific recombination between the <i>attB</i> site of the chromosome and the <i>attP</i> site of the vector. Using this method I have constructed 60 mutation strains of <i>E. coli </i>which contain the trinucleotide repeat tract and are deficient in genes involved in replication, recombination, secondary structure repair or mismatch repair. Techniques for the analysis of the instability of the trinucleotide repeat arrays were developed and used to quantify repeat instability. These included: digestion of chromosomal DNA with a rare-cutting restriction endonuclease and PAGE of the labelled fragments; PCR of the trinucleotide repeat tract, followed by restriction enzyme digestion and PAGE; fluorescent PCR and f-TRAMP (fluorescent trinucleotide amplification which uses just one primer in repeated cycles of linear primer extension): products were separated by capillary electrophoresis and analysed using Gene Scan software. Intensive analyses of different <i>E. coli </i>mutant showed that the trinucleotide repeat arrays integrated into the chromosome are stable. Except in one case, no instability was observed in any mutant deficient in replication, recombination, mismatch repair or secondary structure repair. The only strain, which showed instability, was a <i>mutD</i> mutant (impaired in the proof-reading activity of DNA Polymerase III). Possible explanations for this observation are discussed.

Trinucleotide repeat (TNR) expansions are the cause of over 40 human neurodegenerative diseases, and are linked to DNA damage and base excision repair (BER). We explored the role of DNA damage and BER in modulating TNR instability through analysis of DNA structures, BER protein activities, and reconstitution of repair using human BER proteins and synthesized DNA containing various types of damage. We show that DNA damage and BER can modulate TNR expansions by promoting removal of a TNR hairpin through coordinated activities of BER proteins and cofactors. We found that during repair in a TNR hairpin, coordination between the 5’-flap endonuclease activity of flap endonuclease 1 (FEN1), 3’-5’ exonuclease activity of AP endonuclease 1 (APE1), and activity of DNA ligase I (LIG I) can resolve the double-flap structure produced during BER in the hairpin loop. The resolution of the double-flap structure resulted in hairpin removal and prevention or attenuation of TNR expansions and provides the first evidence that coordination among BER proteins can remove a TNR hairpin. We further explored the role of BER cofactors in modulating TNR instability and found that the repair cofactor proliferating cell nuclear antigen (PCNA) facilitates genomic stability by promoting removal of a TNR hairpin. Hairpin removal was accomplished by altering dynamic TNR structures to allow more efficient FEN1 cleavage and DNA polymerase β (pol β) synthesis and stimulating the activity of LIG I. This study provides the first evidence that a DNA repair cofactor plays an important role in modulating TNR instability. Finally, we explored the effects of sugar modifications in abasic sites on activities of BER proteins and BER efficiency during repair in a TNR tract. We found that an oxidized sugar inhibits the activities of BER enzymes, interrupting their coordination and preventing efficient repair. Inefficient repair results in accumulation of repair intermediates with DNA breaks, contributing to genomic instability. Our results indicate that DNA base lesions and BER play a crucial role in modulating TNR instability. The research presented herein provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

Trinucleotide repeat (TNR) expansion is the cause of more than 40 types of human neurodegenerative diseases such as Huntington’s disease. Recent studies have linked TNR expansion with oxidative DNA damage and base excision repair (BER). In this research, we provided the first evidence that oxidative DNA damage can induce CAG repeat deletion/contraction via BER. We found that BER of an oxidized DNA base lesion, 8-oxoguanine in a CAG repeat tract, resulted in the formation of a CTG hairpin at the template strand. DNA polymerase β (pol b) then skipped over the hairpin creating a 5’-flap that was cleaved by flap endonuclease 1 (FEN1) leading to CAG repeat deletion. To further investigate whether BER may help to shorten an expanded TNR tract, we examined BER in a CAG repeat hairpin loop. We found that 8-oxoguanine DNA glycosylase removed the oxidized base located in the loop region of the hairpin leaving an abasic site. Apurinic/apyrimidinic (AP) endonuclease 1 then incised the 5’-end of the abasic site leaving a nick in the loop. This further converted the hairpin into an intermediate with a 3’-flap and a 5’-flap. As a 5’-3’ endonuclease, FEN1 cleaved the 5’-flap, whereas a 3’-5’ endonuclease, Mus81/Eme1, removed the 3’-flap. The coordination between FEN1 and Mus81/Eme1 ultimately resulted in removal of a CAG repeat hairpin attenuating or preventing TNR expansion. To further explore if pol β bypass of an oxidized base lesion, 5’,8-cyclodeoxyadenosine, may affect TNR instability, we examined pol β DNA synthesis in bypassing this base lesion and found that the lesion preferentially induced TNR deletion during BER and Okazaki fragment maturation. The repeat deletion was mediated by the formation of a loop in the template strand induced specifically by the damage. Pol β then skipped over the loop structure creating a 5’-flap that was efficiently removed by FEN1 leading to repeat deletion. Our study demonstrates that pol β-mediated BER plays an important role in mediating TNR deletion and removing a TNR hairpin to prevent TNR expansion. Our research provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.