Instability Of Repeat Sequences Research Articles

Possibilities and challenges of small molecule organic compounds for the treatment of repeat diseases.

The instability of repeat sequences in the human genome results in the onset of many neurological diseases if the repeats expand above a certain threshold. The transcripts containing long repeats sequester RNA binding proteins. The mechanism of repeat instability involves metastable slip-out hairpin DNA structures. Synthetic organic chemists have focused on the development of small organic molecules targeting repeat DNA and RNA sequences to treat neurological diseases with repeat-binding molecules. Our laboratory has studied a series of small molecules binding to mismatched base pairs and found molecules capable of binding CAG repeat DNA, which causes Huntington’s disease upon expansion, CUG repeat RNA, a typical toxic RNA causing myotonic dystrophy type 1, and UGGAA repeat RNA causing spinocerebellar ataxia type 31. These molecules exhibited significant beneficial effects on disease models in vivo, suggesting the possibilities for small molecules as drugs for treating these neurological diseases.

Integrated analysis sheds light on evolutionary trajectories of young transcription start sites in the human genome.

Understanding the molecular mechanisms and evolution of the gene regulatory system remains a major challenge in biology. Transcription start sites (TSSs) are especially interesting because they are central to initiating gene expression. Previous studies revealed widespread transcription initiation and fast turnover of TSSs in mammalian genomes. Yet, how new TSSs originate and how they evolve over time remain poorly understood. To address these questions, we analyzed ∼200,000 human TSSs by integrating evolutionary (inter- and intra-species) and functional genomic data, particularly focusing on evolutionarily young TSSs that emerged in the primate lineage. TSSs were grouped according to their evolutionary age using sequence alignment information as a proxy. Comparisons of young and old TSSs revealed that (1) new TSSs emerge through a combination of intrinsic factors, like the sequence properties of transposable elements and tandem repeats, and extrinsic factors such as their proximity to existing regulatory modules; (2) new TSSs undergo rapid evolution that reduces the inherent instability of repeat sequences associated with a high propensity of TSS emergence; and (3) once established, the transcriptional competence of surviving TSSs is gradually enhanced, with evolutionary changes subject to temporal (fewer regulatory changes in younger TSSs) and spatial constraints (fewer regulatory changes in more isolated TSSs). These findings advance our understanding of how regulatory innovations arise in the genome throughout evolution and highlight the genomic robustness and evolvability in these processes.

Differences in genome-wide repeat sequence instability conferred by proofreading and mismatch repair defects.

Mutation rates are used to calibrate molecular clocks and to link genetic variants with human disease. However, mutation rates are not uniform across each eukaryotic genome. Rates for insertion/deletion (indel) mutations have been found to vary widely when examined in vitro and at specific loci in vivo. Here, we report the genome-wide rates of formation and repair of indels made during replication of yeast nuclear DNA. Using over 6000 indels accumulated in four mismatch repair (MMR) defective strains, and statistical corrections for false negatives, we find that indel rates increase by 100 000-fold with increasing homonucleotide run length, representing the greatest effect on replication fidelity of any known genomic parameter. Nonetheless, long genomic homopolymer runs are overrepresented relative to random chance, implying positive selection. Proofreading defects in the replicative polymerases selectively increase indel rates in short repetitive tracts, likely reflecting the distance over which Pols δ and ϵ interact with duplex DNA upstream of the polymerase active site. In contrast, MMR defects hugely increase indel mutagenesis in long repetitive sequences. Because repetitive sequences are not uniformly distributed among genomic functional elements, the quantitatively different consequences on genome-wide repeat sequence instability conferred by defects in proofreading and MMR have important biological implications.

DNA Mismatch Repair Defects and Microsatellite Instability Status in Periocular Sebaceous Carcinoma

To characterize mismatch repair protein expression and the role of DNA repair abnormalities in sebaceous carcinomas of the ocular adnexa. Retrospective case-series study. We reviewed 10 cases of sporadic sebaceous carcinoma and 1 case involving a patient with a family history consistent with Muir-Torre syndrome. Immunohistochemistry was used to analyze the presence of 4 mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) in these tumors. DNA was extracted from 7 of the larger tumors as well as from adjacent normal control tissue and microsatellite instability (MSI) analysis using 5 highly sensitive mononucleotides and 2 pentanucleotides was performed. All 10 sporadic periocular sebaceous carcinomas maintained strong staining of the 4 mismatch repair genes, while tumor from the patient with Muir-Torre syndrome showed loss of staining for the mismatch repair genes MSH2 and MSH6. MSI testing of 7 tumors identified no changes in sporadic cases and yielded results supporting presence of repeat sequence instability in the Muir-Torre-associated case. Sporadic sebaceous carcinoma of the ocular adnexa is not commonly associated with a loss of mismatch repair genes or microsatellite instability.

Germ-line CAG repeat instability causes extreme CAG repeat expansion with infantile-onset spinocerebellar ataxia type 2

The spinocerebellar ataxias (SCA) are a genetically and clinically heterogeneous group of diseases, characterized by dominant inheritance, progressive cerebellar ataxia and diverse extracerebellar symptoms. A subgroup of the ataxias is caused by unstable CAG-repeat expansions in their respective genes leading to pathogenic expansions of polyglutamine stretches in the encoded proteins. In general, unstable CAG repeats have an uninterrupted CAG repeat, whereas stable CAG repeats are either short or interrupted by CAA codons, which - like CAG codons - code for glutamine. Here we report on an infantile SCA2 patient who, due to germ-line CAG repeat instability in her father, inherited an extremely expanded CAG repeat in the SCA2 locus. Surprisingly, the expanded allele of the father was an interrupted CAG repeat sequence. Furthermore, analyses of single spermatozoa showed a high frequency of paternal germ-line repeat sequence instability of the expanded SCA2 locus.

Mismatch repair-independent tandem repeat sequence instability resulting from ribonucleotide incorporation by DNA polymerase ɛ

During DNA synthesis in vitro using dNTP and rNTP concentrations present in vivo, yeast replicative DNA polymerases α, δ and ɛ (Pols α, δ and ɛ) stably incorporate rNTPs into DNA. rNTPs are also incorporated during replication in vivo, and they are repaired in an RNase H2-dependent manner. In strains encoding a mutator allele of Pol ɛ ( pol2-M644G), failure to remove rNMPs from DNA due to deletion of the RNH201 gene encoding the catalytic subunit of RNase H2, results in deletion of 2–5 base pairs in short repetitive sequences. Deletion rates depend on the orientation of the reporter gene relative to a nearby replication origin, suggesting that mutations result from rNMPs incorporated during replication. Here we demonstrate that 2–5 base pair deletion mutagenesis also strongly increases in rnh201Δ strains encoding wild type DNA polymerases. As in the pol2-M644G strains, the deletions occur at repetitive sequences and are orientation-dependent, suggesting that mismatches involving misaligned strands arise that could be subject to mismatch repair. Unexpectedly however, 2–5 base pair deletion rates resulting from loss of RNH201 in the pol2-M644G strain are unaffected by concomitant loss of MSH3, MSH6, or both. It could be that the mismatch repair machinery is unable to repair mismatches resulting from unrepaired rNMPs incorporated into DNA by M644G Pol ɛ, but this possibility is belied by the observation that Msh2–Msh6 can bind to a ribonucleotide-containing mismatch. Alternatively, following incorporation of rNMPs by M644G Pol ɛ during replication, the conversion of unrepaired rNMPs into mutations may occur outside the context of replication, e.g., during the repair of nicks resulting from rNMPs in DNA. The results make interesting predictions that can be tested.

Radiation-Induced Genomic Instability in Tandem Repeat Sequences is not Predictive of Unique Sequence Instability

Tandem repeat sequences, classified as minisatellite sequences or partially duplicated genes, are inherently unstable. Radiation exposure can increase the instability of such repeat sequences, but the biological consequences of this elevated instability are not well characterized. To learn more about the characteristics of the instability at different sequences in the genome, we created mutant HT1080 cells bearing 8.4 kb of partially duplicated allele at the HPRT locus by gene targeting. The cells were then tested to determine whether repeat-sequence instability (assessed by elevated reversion rate caused by loss of one duplicated segment) accompanied increased forward mutation rates at the restored wild-type HPRT allele. After a 4-Gy X irradiation, 32 clones were selected (out of 500 clones, 6%) that showed elevated reversion rates even after many cell generations. These clones also showed general increases in the forward mutation rate, whereas the paired individual mutation rates did not correlate with each other. Furthermore, levels of intracellular reactive oxygen species (ROS) and nuclear gamma-H2AX foci, which are hallmarks for DNA damage responses, were also generally elevated, although the levels did not correlate with the individual reversion rates. It was concluded that repeat sequence instability is not predictive of unique sequence instability, probably because the instability is generated by multiple mechanisms after radiation exposure.

Repeat sequence instability is not closely linked to unique sequence instability in X-irradiated HT1080 (HPRTdup) cells

Repeat sequence instability is not closely linked to unique sequence instability in X-irradiated HT1080 (HPRTdup) cells

Infrequent microsatellite instability in liver fluke infection-associated intrahepatic cholangiocarcinomas from Thailand.

The liver fluke infection-associated intrahepatic cholangiocarcinoma (ICC) is a major liver cancer in Northeast Thailand. The molecular basis of this ICC is poorly understood. To address possible roles of the DNA mismatch repair (MMR) system in ICC carcinogenesis, a fluorescence-labeling PCR/laser scanning technique with high sensitivity was employed to analyze genomic instability in the nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in 24 fresh and 13 formalin-fixed, paraffin-embedded tissues of ICC and their corresponding normal parts. Microsatellite instability (MSI) was assessed in nDNA, using 12 highly polymorphic loci including 5 Bethesda markers. These loci were mainly related to major MMR genes, hMSH2 and hMLH1. Also 3 (C)n and/or (C)n(A)n repeat instability at 1 noncoding region in the displacement-loop (D-loop) and 2 coding sequences in NADH dehydrogenase subunit 1 and subunit 5 gene in mtDNA were analyzed. MSI was only detected in 1 (2.7%), 6 (16.7%), 1 (2.9%), 1 (2.9%) or 2 (6.3%) out of 37, 36, 35, 35 or 32 cases at BAT-25, D2S123, D3S1611, D11S904 or D17S250, respectively. LOH was found at D3S1298, D3S1561, D5S346 and TP53 in 4 (18.2%) out of 22, 2 (18.2%) out of 11, 6 (33.3%) out of 18 and 3 (12.5%) out of 24 informative cases, respectively. In mtDNA, none except a single case out of the 37 (2.7%) exhibited repeat sequence instability in the D-loop. We conclude that the liver fluke infection-associated ICC in Thailand is classified as low frequency MSI or microsatellite stable type and that DNA MMR system, through hMSH2 and hMLH1 gene mutations, does not play a major role in its carcinogenesis.

Deletion errors generated during replication of CAG repeats.

Triplet repeat sequence instability is associated with hereditary neurological diseases and with certain types of cancer. Here we study one form of this instability, deletion of triplet repeats during replication of template (CAG)(n)sequences by DNA polymerases. To monitor loss of triplet codons, we inserted (CAG)(9)and (CAG)(17)repeats into the lacZ sequence in M13mp2 and changed one repeat to a TAG codon to yield DNA substrates with colorless plaque phenotypes. Templates containing these inserts within gaps were copied and errors were scored as blue plaque Lac revertants whose DNA was sequenced to determine if loss of the TAG codon resulted from substitutions or deletions. DNA synthesis by either DNA polymerase beta or exonuclease-deficient T7 DNA polymerase produced deletions involving loss of from 1 to 8 of 9 or 15 of 17 repeats. Thus, these polymerases utilize misaligned template-primers containing from 3 to 45 extra template strand nucleotides. Deletion frequencies were much higher than substitution frequencies at the TAG codon in certain repeats, indicating that triplet repeats are at high risk for mutation in the absence of error correction. Proofreading-proficient T7 DNA polymerase generated deletions at 2- to 10-fold lower frequencies than did its exonuclease-deficient derivative. This suggests that misaligned triplet repeat sequences are subject to proofreading, but at reduced efficiency compared to editing of single-base mismatches.

Mutated gene-specific phenotypes of dinucleotide repeat instability in human colorectal carcinoma cell lines deficient in DNA mismatch repair.

Mutations in DNA mismatch repair (MMR) genes in hereditary non-polyposis colon cancer (HNPCC) patients revealed the importance of MMR deficiency as a risk for carcinogenesis. Since diverse mutations occur in several MMR genes, the instability of repeat sequences dispersed in the genome, which are also governed by the MMR system, is a well used marker. However, the relationship between repeat sequence instability and MMR gene mutation in human cells has not been well defined mainly because precise systems to analyse repeat sequences have not been available. Using our newly developed system, we analysed alteration of dinucleotide repeats in human cell lines which harbour mutations in MMR genes. Among 24 subclones of DLD-1 cells (hMSH6-) only one had a dinucleotide repeat alteration in only one microsatellite locus, while LoVo cells (hMSH2-/hMSH6-) exhibited marked dinucleotide repeat instability (DRI). HCT116 cells, a hMLH1-mutant, showed an ultimate DRI phenotype. Interestingly, SW48 cells lacking hMLH1 expression also demonstrated DRI, albeit the extent of diversity being significantly lower than HCT116. These data suggest that the DRI phenotype in human cells is highly dependent on mutated MMR genes and on forms of mutation. The results of DRI analyses used to detect MMR-deficiency should be interpreted with caution.

Apparent protection from instability of repeat sequences in cancer-related genes in replication error positive gastrointestinal cancers.

Genomic instability at simple repeated sequences has been observed in various types of human cancers and is considered an important mechanism in tumorigenesis. Alterations at microsatellite loci have been reported scattered throughout the genome. Recently, the transforming growth factor-beta receptor type II (TGF-beta RII) and the insulin-like growth factor II receptor (IGF-IIR) genes were shown to have inactivating mutations within coding microsatellite sequences. The demonstration of mutations in two growth regulatory genes supports the idea that other regulatory genes with repeat sequences may also be targets in tumours with defective mismatch repair. We examined genes involved in tumour suppression, cell adhesion and cell cycle regulation for mutations at small repeat sequences in replication error positive gastrointestinal cancers. Several polymorphisms were found which exhibited instability, but no other instability was present in the regions examined.

Nucleotide sequence analysis of the apolipoprotein B 3' VNTR

Variable number of tandem repeat (VNTR) loci typically exhibit high rates of germline mutations that alter allele length and thus are ideal models for examining processes governing repeat sequence instability. We have characterized by nucleotide sequencing the internal structure of the apolipoprotein B (Apo B) 3' VNTR in a sample of same- and different-sized alleles previously associated with flanking marker haplotypes. Significant linkage disequilibrium between flanking polymorphisms and minisatellite alleles excludes unequal recombination as the predominant mechanism of mutation at the Apo B VNTR and is consistent with intra-allelic mutational processes such as replication slippage and/or unequal sister chromatid exchange. Diversity among different length alleles was distinctly polar and was usually attributable to changes in copy number at one particular repeat sequence. Analysis of predicted secondary structures for the dimeric repeats demonstrated a relationship between variability and the potential to form self-complementary intermediates. Preferential instability of the variable repeat: (i) was a function of its location within the tandem array; (ii) was not solely dependent on copy number; and (iii) may be related to the base composition of the VNTR and the degree of self-complementarity between the dimeric repeat sequences. The data suggest that polarized variability may be independent of the mutational process(es) generating length variation at minisatellite loci and suggest a possible alternative mechanism of mutation that involves the formation of secondary structures.