Short Repetitive DNA Sequences Research Articles

Telomere length is not a useful tool for chronological age estimation in animals.

Telomeres are short repetitive DNA sequences capping the ends of chromosomes. Telomere shortening occurs during cell division and may be accelerated by oxidative damage or ameliorated by telomere maintenance mechanisms. Consequently, telomere length changes with age, which was recently confirmed in a large meta-analysis across vertebrates. However, based on the correlation between telomere length and age, it was concluded that telomere length can be used as a tool for chronological age estimation in animals. Correlation should not be confused with predictability, and the current data and studies suggest that telomeres cannot be used to reliably predict individual chronological age. There are biological reasons for why there is large individual variation in telomere dynamics, which is mainly due to high susceptibility to a wide range of environmental, but also genetic factors, rendering telomeres unfeasible as a tool for age estimation. The use of telomeres for chronological age estimation is largely a misguided effort, but its occasional reappearance in the literature raises concerns that it will mislead resources in wildlife conservation.

Can the tumor-agnostic evaluation of MSI/MMR status be the common denominator for the immunotherapy treatment of patients with several solid tumors?

Alterations in short-repetitive DNA sequences, known as microsatellite instability (MSI), can reflect deficiencies in Mismatch Repair (MMR) system which represents a major player in DNA integrity maintenance. The incidence of MSI-H/dMMR has been shown to be variable depending on the tumor type. Several studies confirmed that dMMR/MSI status, although less frequent than PD-L1 expression, may better predict response to immune-checkpoint inhibitors (ICIs) in patients with solid tumors. In October 2016, the FDA granted pembrolizumab as breakthrough therapy for the treatment of non-CRC, MSI-H/dMMR tumors, providing, for the first time, a tumor-agnostic indication. In the next future, the tissue-agnostic evaluation of MSI-H/dMMR could become the common denominator for the immunotherapy treatment of patients with different advanced solid tumors, in order to select patient subgroups which may benefit from this therapy. In this Review we provided an overview of the main clinical studies describing the association between MSI-H/dMMR tumors and immunotherapy response.

Loss of MMR and TGFBR2 Increases the Susceptibility to Microbiota-Dependent Inflammation-Associated Colon Cancer.

Background and AimsMutations in DNA mismatch repair (MMR) genes are causative in Lynch syndrome and a significant proportion of sporadic colorectal cancers (CRCs). MMR-deficient (dMMR) CRCs display increased mutation rates, with mutations frequently accumulating at short repetitive DNA sequences throughout the genome (microsatellite instability). The TGFBR2 gene is one of the most frequently mutated genes in dMMR CRCs. Therefore, we generated an animal model to study how the loss of both TGFBR2 signaling impacts dMMR-driven intestinal tumorigenesis in vivo and explore the impact of the gut microbiota.MethodsWe generated VCMsh2/Tgfbr2 mice in which Msh2loxP and Tgfbr2loxP alleles are inactivated by Villin-Cre recombinase in the intestinal epithelium. VCMsh2/Tgfbr2 mice were analyzed for their rate of intestinal cancer development and for the mutational spectra and gene expression profiles of tumors. In addition, we assessed the impact of chemically induced chronic inflammation and gut microbiota composition on colorectal tumorigenesis.ResultsVCMsh2/Tgfbr2 mice developed small intestinal adenocarcinomas and CRCs with histopathological features highly similar to CRCs in Lynch syndrome patients. The CRCs in VCMsh2/Tgfbr2 mice were associated with the presence of colitis and displayed genetic and histological features that resembled inflammation-associated CRCs in human patients. The development of CRCs in VCMsh2/Tgfbr2 mice was strongly modulated by the gut microbiota composition, which in turn was impacted by the TGFBR2 status of the tumors.ConclusionsOur results demonstrate a synergistic interaction between MMR and TGFBR2 inactivation in inflammation-associated colon tumorigenesis and highlight the crucial impact of the gut microbiota on modulating the incidence of inflammation-associated CRCs.

On the comparative biology of mammalian telomeres: Telomere length co-evolves with body mass, lifespan and cancer risk.

Telomeres, the short repetitive DNA sequences that cap the ends of linear chromosomes, shorten during cell division and are implicated in senescence in most species. Telomerase can rebuild telomeres but is repressed in many mammals that exhibit replicative senescence, presumably as a tumour suppression mechanism. It is therefore important to understand the co-evolution of telomere biology and life-history traits that has shaped the diversity of senescence patterns across species. Gomes et al. previously produced a large data set on telomere length (TL), telomerase activity, body mass and lifespan among 57 mammal species. We re-analysed their data using the same phylogenetic multiple regressions and with several additional analyses to test the robustness of the findings. We found substantial inconsistencies in our results compared to Gomes et al.'s. Consistent with Gomes et al. we found an inverse association between TL and lifespan. Contrary to the analyses in Gomes et al., we found a generally robust inverse association between TL and mass, and only weak nonrobust evidence for an association between telomerase activity and mass. These results suggest that shorter TL may have been selected for in larger and longer lived species, probably as a mechanism to suppress cancer. We support this hypothesis by showing that longer telomeres predict higher cancer risk across 22 species. Furthermore, we find that domesticated species have longer telomeres. Our results call into question past interpretations of the co-evolution of telomere biology and life-history traits and stress the need for careful attention to model construction.

Microsatellite Analysis of Geographically Close Isolates of Cystoisospora suis.

Microsatellites are short repetitive DNA sequences of 2–6 repeats interspersed in the genome that display a rapid mutation rate and consequently show high variation between individuals or populations. They have been used to characterize population diversity and structure and the level of variation between different isolates of a number of different organisms, including apicomplexan protozoa. Currently nothing is known about the genetic variability and population structure of Cystoisospora suis (Apicomplexa: Coccidia), the causative agent of piglet coccidiosis, and we made use of the recently available genome of C. suis (strain Wien-I) to amplify microsatellite regions (ca. 300–550 bp) and evaluate the applicability of fluorescence-labeled primers to investigate amplicon length variation at high resolution using capillary electrophoresis (CE). Two phenotypically characterized isolates (Wien-I, toltrazuril susceptible; Holl 1 toltrazuril resistant) and six field isolates from Europe were compared by conventional PCR followed by agar-gel electrophoresis, Sanger sequencing, and CE (fluorescence labeling and fragment length analysis) to evaluate the applicability of the method. Four primer pairs could be identified that amplified bands of the expected size and were labeled for CE analysis. High resolution CE for size determination of PCR amplicons proved to be a reliable and simple method. It revealed high diversity of the analyzed strains, with marked differences even between two strains from neighboring swine farms. In follow-up studies, adaptation of the PCR assay to multiplexing and amplification of small DNA quantities will provide a cost-effective tool to analyse field strains to reveal geographic diversity that could be mapped to phenotypic traits.

DNA mismatch repair in cancer.

Microsatellite instability (MSI) refers to the hypermutator phenotype secondary to frequent polymorphism in short repetitive DNA sequences and single nucleotide substitution, as consequence of DNA mismatch repair (MMR) deficiency. MSI secondary to germline mutation in DNA MMR proteins is the molecular fingerprint of Lynch syndrome (LS), while epigenetic inactivation of these genes is more commonly found in sporadic MSI tumors. MSI occurs at different frequencies across malignancies, although original methods to assess MSI or MMR deficiency have been developed mostly in LS related cancers. Here we will discuss the current methods to detect MSI/MMR deficiency with a focus of new tools which are emerging as highly sensitive detector for MSI across multiple tumor types. Due to high frequencies of non-synonymous mutations, the presence of frameshift-mutated neoantigens, which can trigger a more robust and long-lasting immune response and strong TIL infiltration with tumor eradication, MSI has emerged as an important predictor of sensitivity for immunotherapy-based strategies, as showed by the recent FDA's first histology agnostic-accelerated approval to immune checkpoint inhibitors for refractory, adult and pediatric, MMR deficient (dMMR) or MSI high (MSI-H) tumors. Moreover, it is known that MSI status may predict cancer response/resistance to certain chemotherapies. Here we will describe the complex interplay between the genetic and clinical-pathological features of MSI/dMMR tumors and the cancer immunotherapy, with a focus on the predictive and prognostic role of MMR status for immune checkpoint inhibitors (ICIs) and providing some suggestions on how to conceive better predictive markers for immunotherapy in the next future.

TGFBR2-dependent alterations of exosomal cargo and functions in DNA mismatch repair-deficient HCT116 colorectal cancer cells

BackgroundColorectal cancers (CRCs) that lack DNA mismatch repair function exhibit the microsatellite unstable (MSI) phenotype and are characterized by the accumulation of frameshift mutations at short repetitive DNA sequences (microsatellites). These tumors recurrently show inactivating frameshift mutations in the tumor suppressor Transforming Growth Factor Beta Receptor Type 2 (TGFBR2) thereby abrogating downstream signaling. How altered TGFBR2 signaling affects exosome-mediated communication between MSI tumor cells and their environment has not been resolved. Here, we report on molecular alterations of exosomes shed by MSI cells and the biological response evoked in recipient cells.MethodsExosomes were isolated and characterized by electron microscopy, nanoparticle tracking, and western blot analysis. TGFBR2-dependent effects on the cargo and functions of exosomes were studied in a MSI CRC model cell line enabling reconstituted and inducible TGFBR2 expression and signaling. Microsatellite frameshift mutations in exosomal and cellular DNA were examined by PCR-based DNA fragment analysis and exosomal protein profiles were identified by mass spectrometry. Uptake of fluorescent-labeled exosomes by hepatoma recipient cells was monitored by confocal microscopy. TGFBR2-dependent exosomal effects on secreted cytokine levels of recipient cells were analyzed by Luminex technology and ELISA.ResultsFrameshift mutation patterns in microsatellite stretches of TGFBR2 and other MSI target genes were found to be reflected in the cargo of MSI CRC-derived exosomes. At the proteome level, reconstituted TGFBR2 expression and signaling uncovered two protein subsets exclusively occurring in exosomes derived from TGFBR2-deficient (14 proteins) or TGFBR2-proficient (five proteins) MSI donor cells. Uptake of these exosomes by recipient cells caused increased secretion (2–6 fold) of specific cytokines (Interleukin-4, Stem Cell Factor, Platelet-derived Growth Factor-B), depending on the TGFBR2 expression status of the tumor cell.ConclusionOur results indicate that the coding MSI phenotype of DNA mismatch repair-deficient CRC cells is maintained in their exosomal DNA. Moreover, we uncovered that a recurrent MSI tumor driver mutation like TGFBR2 can reprogram the protein content of MSI cell-derived exosomes and in turn modulate the cytokine secretion profile of recipient cells. Apart from its diagnostic potential, these TGFBR2-dependent exosomal molecular and proteomic signatures might help to understand the signaling routes used by MSI tumors.Graphical Fricke et al. uncovered coding microsatellite instability-associated mutations of colorectal tumor driver genes like TGFBR2 in MSI tumor cellderived exosomes. Depending on the TGFBR2 expression status of their donor cells, shed exosomes show distinct proteomic signatures and promote altered cytokine secretion profiles in recipient cells.

Streptococcus pneumoniae Genome-wide Identification and Characterization of BOX Element-binding Domains.

The BOX elements are short repetitive DNA sequences that distribute randomly in intergenic regions of the Streptococcus pneumoniae genome. The function and origin of such elements are still unknown, but they were found to modulate expression of neighboring genes. Evidences suggested that the modulation's mechanism can be fulfilled by sequence-specific interaction of BOX elements with transcription factor family proteins. However, the type and function of these BOX-binding proteins still remain largely unexplored to date. In the current study we described a synthetic protocol to investigate the recognition and interaction between a highly conserved site of BOX elements and the DNA-binding domains of a variety of putative transcription factors in the pneumococcal genome. With the protocol we were able to predict those high-affinity domain binders of the conserved BOX DNA site (BOX DNA) in a high-throughput manner, and analyzed sequence-specific interaction in the domainDNA recognition at molecular level. Consequently, a number of putative transcription factor domains with both high affinity and specificity for the BOX DNA were identified, from which the helix-turn-helix (HTH) motif of a small heat shock factor was selected as a case study and tested for its binding capability toward the double-stranded BOX DNA using fluorescence anisotropy analysis. As might be expected, a relatively high affinity was detected for the interaction of HTH motif with BOX DNA with dissociation constant at nanomolar level. Molecular dynamics simulation, atomic structure examination and binding energy analysis revealed a complicated network of intensive nonbonded interactions across the complex interface, which confers both stability and specificity for the complex architecture.

Estrogen pathway mutations and cancer

Cancer is caused by an accumulation of mutations in a stem cell. A defective mismatch repair (MMR) system can lead to such an accumulation of mutations. MMR defects are found in a cancer syndrome called Lynch Syndrome, and tumors of this syndrome are indeed characterized by such an accumulation of mutations, particularly in short repetitive DNA sequences, called microsatellites. When such mutated microsatellites are located in the coding sequences of genes with essential roles for tumorigenesis, we speak of ‘target genes’. Many such target genes have been found and in this review we focus the possible involvement of target genes involved in the estrogen-receptor pathway (ER). We recently identified NRIP1, encoding the nuclear receptor-interacting protein 1, as the most frequently mutated gene in microsatellite instable (MSI) endometrial cancer (EC). NRIP1 is a known corepressor of the ER pathway, the pathway essential in regulating the concentrations of estrogens, a hormone for which the endometrium is highly responsive. This in combination with the fact that high exposure to estrogens is currently considered the major risk factor for EC - approximately 80% of all sporadic EC tumors are estrogen dependent carcinomas - make NRIP1 the perfect target gene for EC. Interestingly, mutations in NRIP1 were also detected in MSI colorectal carcinoma (CRC) samples. Finding mutations in an estrogen receptor signaling protein in colorectal tissue might not be that expected, as colon is not typically associated with being responsive to estrogens. However, evidence is accumulating to better understand this finding. For instance, it was shown that NRIP1 , in colon tissue, stimulates APC gene transcription and inhibits β-catenin activation. Moreover, some studies suggested that estrogens can increase the expression of MLH1 in colon cancer cells, highlighting the implications of estrogen protecting against colon cancer, by regulating the MMR system. All in all we conclude that genes involved in the estrogen pathway are the perfect candidates to be studied in MMR-deficient tumors, especially those developing in hormonal responsive tissues.

Trastuzumab-induced HER2 phosphorylation: exploring the mechanisms and implications

Cancer is caused by an accumulation of mutations in a stem cell. A defective mismatch repair (MMR) system can lead to such an accumulation of mutations. MMR defects are found in a cancer syndrome called Lynch Syndrome, and tumors of this syndrome are indeed characterized by such an accumulation of mutations, particularly in short repetitive DNA sequences, called microsatellites. When such mutated microsatellites are located in the coding sequences of genes with essential roles for tumorigenesis, we speak of ‘target genes’. Many such target genes have been found and in this review we focus the possible involvement of target genes involved in the estrogen-receptor pathway (ER). We recently identified NRIP1, encoding the nuclear receptor-interacting protein 1, as the most frequently mutated gene in microsatellite instable (MSI) endometrial cancer (EC). NRIP1 is a known corepressor of the ER pathway, the pathway essential in regulating the concentrations of estrogens, a hormone for which the endometrium is highly responsive. This in combination with the fact that high exposure to estrogens is currently considered the major risk factor for EC - approximately 80% of all sporadic EC tumors are estrogen dependent carcinomas - make NRIP1 the perfect target gene for EC. Interestingly, mutations in NRIP1 were also detected in MSI colorectal carcinoma (CRC) samples. Finding mutations in an estrogen receptor signaling protein in colorectal tissue might not be that expected, as colon is not typically associated with being responsive to estrogens. However, evidence is accumulating to better understand this finding. For instance, it was shown that NRIP1, in colon tissue, stimulates APC gene transcription and inhibits β-catenin activation. Moreover, some studies suggested that estrogens can increase the expression of MLH1 in colon cancer cells, highlighting the implications of estrogen protecting against colon cancer, by regulating the MMR system. All in all we conclude that genes involved in the estrogen pathway are the perfect candidates to be studied in MMR-deficient tumors, especially those developing in hormonal responsive tissues.

Distribution of TTAGG-specific telomerase activity in insects.

In most eukaryotes, telomeres consist of tandem arrays of a short repetitive DNA sequence. Insect telomeres are generally constituted by a (TTAGG)n repeat motif. Usually, telomeres are maintained by telomerase, a specialized reverse transcriptase that adds this sequence to chromosome ends. We examined telomerase activity in 15 species across Insecta. Telomerase activity was revealed in Isoptera, Blattaria, Lepidoptera, Hymenoptera, Trichoptera, Coleoptera, and Sternorrhyncha. In contrast, we were not able to detect telomerase activity in Orthoptera, Zygentoma, and Phasmida. Because we found telomerase activity in phylogenetically distant species, we conclude that a distribution pattern of (TTAGG)n sequence in Insecta is generally consistent with that of telomerase activity. Thus, the TTAGG-telomerase system is functional across the Insecta. Using real-time quantitative telomeric repeat amplification protocol (RTQ-TRAP) system, we quantified telomerase activity in different developmental stages and different tissues of a cockroach, Periplaneta americana. We show that telomerase is upregulated in young instars and gradually declines during development. In adults, it is most active in testes and ovaries. Thus, the telomerase activity of hemimetabolous insects seems to be associated with cell proliferation and organismal development.

Analyzing the Relationship between Single Base Flipping and Strand Slippage near DNA Duplex Termini

Insertion-deletion (indel) mutations are caused by strand slippage between pairing primer and template strands during nucleic acid strand extension. A possible causative factor for such strand slippage is base flipping in the primer strand or template strand, for insertion or deletion mutations, respectively. A simple mechanistic description is that the "hole" in the nucleic acid duplex left behind by a flipping base is occupied by a neighboring base on the same strand, resulting in slippage with respect to its paired strand. The extent of single base flipping required for occupation of its former place in the double helix by a neighboring base is not fully understood. The present study uses restrained molecular dynamics (MD) simulations along a pseudohedihedral base flipping parameter to construct two-dimensional free energy profiles along base flipping and strand slippage geometric parameters. These profiles, generated for both cytosine and guanine single base flipping in a short repetitive indel mutation hot-spot DNA sequence, illustrate the extent of single base flipping that can allow strand slippage by one base position. Relatively minor base flipping into both the major and minor grooves can result in strand slippage. Deconstruction of the collective variable strand slippage geometric parameter into its component distances illustrates the details of how strand slippage can accompany base flipping. The trans Watson-Crick:sugar edge interaction that stabilizes cytosine flipping in this hot-spot sequence is also characterized energetically. The impact of these results on understanding sequence dependence of indel errors in nucleic acid strand extension is discussed, along with a suggestion for future studies that can generalize the present findings to all nearest-neighbor sequence contexts.

Abstract CN06-02: Insight into colorectal cancer etiology through MPE: Unmasking the links with obesity and smoking

Abstract Colorectal cancer is a leading cause of cancer morbidity and mortality worldwide. Epidemiologic studies have identified a host of genetic, lifestyle and demographic factors associated with colorectal cancer incidence, including high body mass index (BMI) and, perhaps less convincingly, cigarette smoking. Epidemiologic studies have often considered colorectal cancer as a singular disease outcome, ignoring potential disease heterogeneity; more recently, however, studies have further investigated whether risk and prognostic factors for colorectal cancer differ by tumor molecular phenotype, including microsatellite instability (MSI) status. MSI is the result of defective DNA mismatch repair capacity and it is a distinct tumor phenotype whereby short repetitive DNA sequences undergo a change in repeat length. Approximately 15% of colorectal tumors display MSI, usually the result of epigenetic modification of MLH1, a gene that encodes a mismatch repair protein, or less often, because the affected individual has a germline mutation in a DNA mismatch repair gene, consistent with Lynch syndrome. The majority of colorectal tumors develop despite having competent mismatch repair; these tumors, known as microsatellite (MS)-stable, arise through chromosomal instability and do not display marked gains or losses in MS regions. Other important molecular phenotypes for colorectal cancer include CpG island methylator phenotype (CIMP), and presence or absence of somatic mutations in the KRAS and BRAF genes. While BMI is a consistent and convincing risk factor for colorectal cancer overall (odds ratios and relative risks in the range of 1.3 to 1.5 are common for an obese BMI relative to a normal BMI), recent studies have suggested the associations are higher, or even restricted to, tumors that are MS-stable. Using the Colon Cancer Family Registry, our group showed that an obese BMI (>30 kg/m^2) relative to a normal BMI (BMI: 18.5-24.99 kg/m^2) was associated with an approximate 2-fold increased risk of MS-stable colorectal cancer while the association with MSI-high colorectal cancer was null. Other recent studies have shown a similar pattern, and other groups have shown that these results might be corroborated with FASN. Current work in the Colon Cancer Family Registry is seeking to address whether the prognostic (i.e., survival) benefit of MSI-high compared to MS-stable tumors further differs by level of obesity. The link between colorectal cancer and cigarette smoking has been more equivocal, with studies generally showing null associations or only modestly higher risks among smokers compared to non-smokers. Herein, tumor molecular phenotype data have been particularly revealing. Data from the Colon Cancer Family Registry showed a null association between ever smoking and colorectal cancer risk overall; however, when stratified by MSI status, risk of MSI-high cancer was about 2-fold higher among long-duration smokers relative to never smokers. Similar findings were reported in the prospective Iowa Women's Health Study where doubling in risks of MSI-high, CIMP-positive and BRAF mutation-positive colorectal cancers were reported for ever smoking while null associations were shown for tumors that lacked these molecular phenotypes. Additional work from one study center involved in the Colon Cancer Family Registry has suggested that the adverse impact of smoking on colorectal cancer survival is limited to patients diagnosed with MSI-high tumors. In addition to presenting published and unpublished results on associations of BMI and smoking with colorectal cancer incidence and survival, this presentation will also share recent experiences with collecting tumor blocks from a prospective cohort study, the American Cancer Society's Cancer Prevention Study-II. Citation Format: Peter T. Campbell. Insight into colorectal cancer etiology through MPE: Unmasking the links with obesity and smoking. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr CN06-02.

127 Template-switching during DNA replication

Short repetitive DNA sequences are frequent sites of genomic rearrangements. Using the bacterium Escherichia coli, we have studied the mechanisms of these frequent events and have proposed that they arise by a template-switch mechanism during DNA replication. We hypothesize that the template-switch mechanism is a replication-gap filling repair mechanism, which, like translesion DNA synthesis, can overcome replication-blocking lesions on the DNA template strand. Both translesion synthesis (TLS) and template-switching are components of what has been termed “post-replication repair.” Tranlesion synthesis has been considered “error-prone” because of its propensity to induce point mutations because of the low fidelity of the TLS polymerases. Template-switching, we propose, comprises what has been considered the “error-free” pathway of post-replication repair. We note that the pathway is not truly error-free, as it is “rearrangement-prone.” Using genetic assays for deletions or expansions at short repeats, we have investigated the properties of the template-switch pathway. Our analysis shows that template-switching frequently occurs, concomitant with sister-chromosome exchange. These crossovers occur independently of the known homologous recombination machinery including RecA. Whereas homologous recombination requires a threshold homology of about 250 bp (below which recombination is inefficient), the template-switching crossovers can occur between microhomologies as low as 25 bp. Blocking replication with the chain-terminator azidothymidine (AZT) stimulates template-switching as do mutations in the DNA polymerase III replisome, consistent with the idea that template-switching occurs as a response to replication gaps. We have identified three proteins whose functions are required for template-switching. Mutants in these functions exhibit enhanced sensitivity to DNA replication inhibitors AZT and hydroxyurea and lower rates of genetic rearrangements. The first template-switch factor is chaperone DnaKJ, identified through a genetic screen. Mutations in dnaK also block the “error-prone” repair pathway, as they exhibit lower rates of DNA polymerase V-dependent mutagenesis induced by UV irradiation. Mutations in the processivity clamp for replication (dnaN) and in one subunit of the clamp loader complex (dnaX) also diminish template-switching. By a programmed translational frameshift, the dnaX gene produces two variants, a larger form required for replication and a shorter form of unknown function. Our recent experiments suggest that the shorter form is required for template-switching. We propose that this short form is used in an alternative clamp loader complex employed during DNA repair.

Regulation of telomere length and homeostasis by telomerase enzyme processivity

Telomerase is a ribonucleoprotein consisting of a catalytic subunit, the telomerase reverse transcriptase (TERT), and an integrally associated RNA that contains a template for the synthesis of short repetitive G-rich DNA sequences at the ends of telomeres. Telomerase can repetitively reverse transcribe its short RNA template, acting processively to add multiple telomeric repeats onto the same DNA substrate. The contribution of enzyme processivity to telomere length regulation in human cells is not well characterized. In cancer cells, under homeostatic telomere length-maintenance conditions, telomerase acts processively, whereas under nonequilibrium conditions, telomerase acts distributively on the shortest telomeres. To investigate the role of increased telomerase processivity on telomere length regulation in human cells with limited lifespan that are dependent on human TERT for lifespan extension and immortalization, we mutated the leucine at position 866 in the reverse transcriptase C motif of human TERT to a tyrosine (L866Y), which is the amino acid found at the equivalent position in HIV-1 reverse transcriptase. We report that, similar to the previously reported gain-of-function Tetrahymena telomerase mutant (L813Y), the human telomerase variant displays increased processivity. Human TERT-L866Y, like wild-type human TERT, can immortalize and extend the lifespan of limited-lifespan cells. Moreover, cells expressing human TERT-L866Y display heterogenous telomere lengths, telomere elongation, multiple telomeric signals indicative of fragile sites and replicative stress, and an increase in short telomeres, which is accompanied by telomere trimming events. Our results suggest that telomere length and homeostasis in human cells may be regulated by telomerase enzyme processivity.

Identification of an MSI-H Tumor-Specific Cytotoxic T Cell Epitope Generated by the (−1) Frame ofU79260(FTO)

Microsatellite instability (MSI-H) induced by defects of the DNA mismatch repair system results in insertion or deletion of single nucleotides at short repetitive DNA sequences. About 15% of sporadic and approximately 90% of hereditary nonpolyposis colorectal cancers display MSI-H. When affecting coding regions, MSI-H results in frameshift mutations and expression of corresponding frameshift peptides (FSPs). Functional tumor promoting relevance has been demonstrated for a growing number of genes frequently hit by MSI-H. Contrary, immune reactions against FSPs are involved in the immune surveillance of MSI-H cancers. Here, we provide conclusive data that the (−1) frame of U79260(FTO) encodes an HLA-A0201-restricted cytotoxic T cell epitope (FSP11; TLSPGWSAV). T cells specific for FSP11 efficiently recognized HLA-A0201(pos) tumor cells harboring the mutated reading frame. Considering the exceptionally high mutation rate of U79260(FTO) in MSI-H colorectal carcinoma (81.8%), this recommends that FSP11 be a component of future vaccines.

Rapid genomic DNA extraction (RGDE)

Hyperpolymorphic short tandem repetitive DNA sequences, STRs or microsatellites, have become widely used in human identification, particularly in criminal cases and in mass disasters. In such cases the substrates for the analyses may be decomposed as a biological material, a fact that has to be taken into account when choosing the appropriate casework methods. Nowadays expanded windows have been opened to the world especially in the area of genetic and biology science by performance of big projects such as human genome project. In this regard, one of the primary and important steps for all is DNA extraction with high quality and quantity in minimum time from biological material. By using RGDE method, genomic DNA with high quality and quantity can be acquired in the shortest time which has been presented in the world up to now. In this paper we report the evaluation of DNA extraction in this method.

T25 Repeat in the 3′ Untranslated Region of the CASP2 Gene: A Sensitive and Specific Marker for Microsatellite Instability in Colorectal Cancer

DNA mismatch repair deficiency is observed in about 10% to 15% of all colorectal carcinomas and in up to 90% of hereditary nonpolyposis colorectal cancer (HNPCC) patients. Tumors with mismatch repair defects acquire mutations in short repetitive DNA sequences, a phenomenon termed high-level microsatellite instability (MSI-H). The diagnosis of MSI-H in colon cancer is of increasing relevance, because MSI-H is an independent prognostic factor in colorectal cancer, seems to influence the efficacy of adjuvant chemotherapy, and is the most important molecular screening tool to identify HNPCC patients. To make MSI typing feasible for the routine pathology laboratory, highly reproducible and cost effective laboratory tests are required. Here, we describe a novel T25 mononucleotide marker in the 3'untranslated region of the CASP2 gene (CAT25) that displayed a quasimonomorphic repeat pattern in normal tissue of 200 unrelated individuals of Caucasian origin. In addition, CAT25 was monomorphic also in all tested donors of African and Asian origin (n = 102 and n = 79, respectively) and thus differs from the most commonly used markers BAT25 and BAT26. Without the analysis of corresponding normal tissue, CAT25 correctly detected 56 of 57 colorectal cancer specimens classified as MSI-H by using the standard National Cancer Institute/International Collaborative Group-HNPCC marker panel. Combined with the standard markers BAT25 and BAT26 in a multiplex PCR, all MSI-H colorectal cancer samples were typed correctly. No false-positive results were obtained in 60 non-MSI-H control colorectal cancer specimens. These data suggest that CAT25 should be included into novel marker panels for microsatellite testing thus allowing for a significant reduction of the complexity and costs of MSI typing. Moreover, CAT25 represents a highly promising marker for early detection of colorectal cancer in HNPCC germ line mutation carriers.

Microsatellite instability and its relevance to cutaneous tumorigenesis.

Increasing evidence suggests that human tumors sequentially accumulate multiple mutations that cannot be explained by the low rates of spontaneous mutations in normal cells (2-3 mutations/cell). The mathematical models estimate that for the solid tumors to develop, as many as 6-12 mutations are required in each tumor cell. Therefore, to account for such high mutation rates, it is proposed that tumor cells are genetically unstable, i.e. they have genome-wide mutations at short repetitive DNA sequences called microsatellites. Microsatellite repeats are scattered throughout the human genome, primarily in the non-coding regions, and can give rise to variants with increased or reduced lengths, i.e. microsatellite instability (MSI). This instability has been reported in an increasing number of cutaneous tumors including: melanocytic tumors, basal cell carcinomas and primary cutaneous T-cell lymphomas. Moreover, MSI has been observed in skin tumors arising in the context of some hereditary disorders such as Muir-Torre syndrome, Von Recklinghausen's disease and disseminated superficial porokeratosis. While MSI in some of these disorders reflects underlying DNA replication errors, the mechanism of instability in others is still unknown. Thus far, MSI is considered to be a distinct tumorigenic pathway that reveals surprising versatility. The ramifications for cutaneous neoplasms warrant further investigation.

Kangaroo – A pattern-matching program for biological sequences

BackgroundBiologists are often interested in performing a simple database search to identify proteins or genes that contain a well-defined sequence pattern. Many databases do not provide straightforward or readily available query tools to perform simple searches, such as identifying transcription binding sites, protein motifs, or repetitive DNA sequences. However, in many cases simple pattern-matching searches can reveal a wealth of information. We present in this paper a regular expression pattern-matching tool that was used to identify short repetitive DNA sequences in human coding regions for the purpose of identifying potential mutation sites in mismatch repair deficient cells.ResultsKangaroo is a web-based regular expression pattern-matching program that can search for patterns in DNA, protein, or coding region sequences in ten different organisms. The program is implemented to facilitate a wide range of queries with no restriction on the length or complexity of the query expression. The program is accessible on the web at http://bioinfo.mshri.on.ca/kangaroo/ and the source code is freely distributed at http://sourceforge.net/projects/slritools/.ConclusionA low-level simple pattern-matching application can prove to be a useful tool in many research settings. For example, Kangaroo was used to identify potential genetic targets in a human colorectal cancer variant that is characterized by a high frequency of mutations in coding regions containing mononucleotide repeats.