Non-repetitive Sequences Research Articles

Construction and Analysis of Suppression Subtractive cDNA Library from Fertile Disk Floret Buds of Zinnia elegans

In order to isolate genes related to anther development and understand molecular basis of male sterility, cDNA library of Zinnia elegans was constructed using suppression subtractive hybridization (SSH) approach. 672 different expressed clones were selected from the fertile disk floret buds. PCR results showed that cDNA inserts were ranged from 100 to 750 bp. 303 positive clones screened by dot-blot hybridization were sequenced. 273 out of 303 sequenced clones produced readable sequences; these sequences represent 87 non-repetitive sequences. The homology alignment showed that 76 expressed sequence tags (ESTs) had functional annotations in GenBank, the other 11 ESTs without any homology to the known gene. In addition, 87 ESTs were divided into 17 groups according to MIPS of Arabidopsis thaliana database. Sequence data from the cDNA library have been deposited with the GenBank under the accession numbers GT067016-GT067085. As an important result in this study, 7 genes related to anther development were isolated. Results from semi-quantitative RT-PCR showed 6 genes were expressed only in disk florets of fertile plants compared with that of male sterile plants. These ESTs obtained provide important clues for further isolation and identification of fertility-related genes in Z. elegans.

Complete mitochondiral genome of the Korean red fox Vulpes vulpes (Carnivora, Canidae)

The complete mitochondrial genome sequence of Vulpes vulpes consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region (CR). CR is located between the tRNA-Pro and tRNA-Phe genes and is 1173 base pairs (bp) in length. It consists of a short non-repetitive sequence followed by 8-bp 5′-ACACACGT-3′ tandem repeat between conserved sequence black I and conserved sequence black II.

Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues

We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines. These microDNAs are 200 to 400 base pairs long, are derived from unique nonrepetitive sequence, and are enriched in the 5'-untranslated regions of genes, exons, and CpG islands. Chromosomal loci that are enriched sources of microDNA in the adult brain are somatically mosaic for microdeletions that appear to arise from the excision of microDNAs. Germline microdeletions identified by the "Thousand Genomes" project may also arise from the excision of microDNAs in the germline lineage. We have thus identified a previously unknown DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci.

New approach to nonrepetitive sequences

AbstractA sequence is nonrepetitive if it does not contain two adjacent identical blocks. The remarkable construction of Thue asserts that three symbols are enough to build an arbitrarily long nonrepetitive sequence. It is still not settled whether the following extension holds: for every sequence of three‐element sets L1,…,Ln there exists a nonrepetitive sequence s1,…,sn with si∈Li. We propose a new non‐constructive way to build long nonrepetitive sequences and provide an elementary proof that sets of size 4 suffice confirming the best known bound. The simple double counting in the heart of the argument is inspired by the recent algorithmic proof of the Lovász local lemma due to Moser and Tardos. Furthermore we apply this approach and present game‐theoretic type results on nonrepetitive sequences. Nonrepetitive game is played by two players who pick, one by one, consecutive terms of a sequence over a given set of symbols. The first player tries to avoid repetitions, while the second player, in contrast, wants to create them. Of course, by simple imitation, the second player can force lots of repetitions of size 1. However, as proved by Pegden, there is a strategy for the first player to build an arbitrarily long sequence over 37 symbols with no repetitions of size greater than 1. Our techniques allow to reduce 37–6. Another game we consider is the erase‐repetition game. Here, whenever a repetition occurs, the repeated block is immediately erased and the next player to move continues the play. We prove that there is a strategy for the first player to build an arbitrarily long nonrepetitive sequence over 8 symbols. © 2012 Wiley Periodicals, Inc. Random Struct. Alg., 2012

Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and β on normal and repetitive DNA sequences

‘Classical’ non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols β, λ and μ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol β, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol β. We propose a possible novel role of DNA pol β in MMEJ, promoting (CAG)n triplet repeats instability.

The Complete Mitochondrial Genome ofCtenoptilum vasava(Lepidoptera: Hesperiidae: Pyrginae) and Its Phylogenetic Implication

We here report the first complete mitochondrial (mt) genome of a skipper, Ctenoptilum vasava Moore, 1865 (Lepidoptera: Hesperiidae: Pyrginae). The mt genome of the skipper is a circular molecule of 15,468 bp, containing 2 ribosomal RNA genes, 24 putative transfer RNA (tRNA), genes including an extra copy of trnS (AGN) and a tRNA-like insertion trnL (UUR), 13 protein-coding genes and an AT-rich region. All protein-coding genes (PCGs) are initiated by ATN codons and terminated by the typical stop codon TAA or TAG, except for COII which ends with a single T. The intergenic spacer sequence between trnS (AGN) and ND1 genes also contains the ATACTAA motif. The AT-rich region of 429 bp is comprised of nonrepetitive sequences, including the motif ATAGA followed by an 19 bp poly-T stretch, a microsatellite-like (AT)3 (TA)9 element next to the ATTTA motif, an 11 bp poly-A adjacent to tRNAs. Phylogenetic analyses (ML and BI methods) showed that Papilionoidea is not a natural group, and Hesperioidea is placed within the Papilionoidea as a sister to ((Pieridae + Lycaenidae) + Nymphalidae) while Papilionoidae is paraphyletic to Hesperioidea. This result is remarkably different from the traditional view where Papilionoidea and Hesperioidea are considered as two distinct superfamilies.

Maternal gametic transmission of translocations or inversions of human chromosome 11p15.5 results in regional DNA hypermethylation and downregulation of CDKN1C expression

Beckwith–Wiedemann syndrome (BWS) is an overgrowth syndrome associated with genetic or epigenetic alterations in one of two imprinted domains on chromosome 11p15.5. Rarely, chromosomal translocations or inversions of chromosome 11p15.5 are associated with BWS but the molecular pathophysiology in such cases is not understood. In our series of 3 translocation and 2 inversion patients with BWS, the chromosome 11p15.5 breakpoints map within the centromeric imprinted domain, 2. We hypothesized that either microdeletions/microduplications adjacent to the breakpoints could disrupt genomic sequences important for imprinted gene regulation. An alternate hypothesis was that epigenetic alterations of as yet unknown regulatory DNA sequences, result in the BWS phenotype. A high resolution Nimblegen custom microarray was designed representing all non-repetitive sequences in the telomeric 33 Mb of the short arm of human chromosome 11. For the BWS-associated chromosome 11p15.5 translocations and inversions, we found no evidence of microdeletions/microduplications. DNA methylation was also tested on this microarray using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. This high-resolution DNA methylation microarray analysis revealed a gain of DNA methylation in the translocation/inversion patients affecting the p-ter segment of chromosome 11p15, including both imprinted domains. BWS patients that inherited a maternal translocation or inversion also demonstrated reduced expression of the growth suppressing imprinted gene, CDKN1C in Domain 2. In summary, our data demonstrate that translocations and inversions involving imprinted domain 2 on chromosome 11p15.5, alter regional DNA methylation patterns and imprinted gene expression in cis, suggesting that these epigenetic alterations are generated by an alteration in “chromatin context”.

Nonrepetitive Sequences on Arithmetic Progressions

A sequence $S=s_{1}s_{2}\ldots s_{n}$ is said to be nonrepetitive if no two adjacent blocks of $S$ are identical. In 1906 Thue proved that there exist arbitrarily long nonrepetitive sequences over $3$-element set of symbols. We study a generalization of nonrepetitive sequences involving arithmetic progressions. We prove that for every $k\geqslant 1$, there exist arbitrarily long sequences over at most $2k+10 \sqrt{k}$ symbols whose subsequences, indexed by arithmetic progressions with common differences from the set $\{1,2,\ldots ,k\}$, are nonrepetitive. This improves a previous bound of $e^{33}k$ obtained by Grytczuk. Our approach is based on a technique introduced recently by Grytczuk Kozik and Micek, which was originally inspired by a constructive proof of the Lovász Local Lemma due to Moser and Tardos. We also discuss some related problems that can be attacked by this method.

Sequencing of BAC pools by different next generation sequencing platforms and strategies

BackgroundNext generation sequencing of BACs is a viable option for deciphering the sequence of even large and highly repetitive genomes. In order to optimize this strategy, we examined the influence of read length on the quality of Roche/454 sequence assemblies, to what extent Illumina/Solexa mate pairs (MPs) improve the assemblies by scaffolding and whether barcoding of BACs is dispensable.ResultsSequencing four BACs with both FLX and Titanium technologies revealed similar sequencing accuracy, but showed that the longer Titanium reads produce considerably less misassemblies and gaps. The 454 assemblies of 96 barcoded BACs were improved by scaffolding 79% of the total contig length with MPs from a non-barcoded library.Assembly of the unmasked 454 sequences without separation by barcodes revealed chimeric contig formation to be a major problem, encompassing 47% of the total contig length. Masking the sequences reduced this fraction to 24%.ConclusionOptimal BAC pool sequencing should be based on the longest available reads, with barcoding essential for a comprehensive assessment of both repetitive and non-repetitive sequence information. When interest is restricted to non-repetitive regions and repeats are masked prior to assembly, barcoding is non-essential. In any case, the assemblies can be improved considerably by scaffolding with non-barcoded BAC pool MPs.

Identification of hornet silk gene with a characteristic repetitive sequence in Vespa simillima xanthoptera

Vssilk 5 is a gene encoding a component protein of the silk produced by the larvae of the yellow hornet (Vespa simillima, Vespinae, Vespidae). In this study, we deduced the complete cDNA sequence of Vssilk 5. It was found that 2 silk proteins, Vssilk 5 N and Vssilk 5 C, in the cocoon of the yellow hornet are both encoded by the Vssilk 5 gene. Vssilk 5 N and 5 C are the N- and C-terminal regions, respectively, of the Vssilk 5 pro-protein (Vssilk 5p). The complete amino acid sequences of Vssilk 5 N and Vssilk 5 C were deduced. Although a non-repetitive amino acid sequence and coiled-coil structure are properties common to the major components of silk proteins produced by the larvae of the social superfamilies Apoidea and Vespoidea of the Apocrita, nearly the entire sequence of Vssilk 5 C consisted of a repeated sequence of amino acids, and the calculated coiled-coil probability for this protein was low. Vssilk 5 N is a protein without a repetitive amino acid sequence and has a low coiled-coil probability. Moreover, we found a water soluble protein, Vssilk 5S that is likely segmented from Vssilk 5 C and contains an N-terminal sequence identical to that of Vssilk 5 C.

Analysis of BAC-end sequences in rainbow trout: Content characterization and assessment of synteny between trout and other fish genomes

BackgroundRainbow trout (Oncorhynchus mykiss) are cultivated worldwide for aquaculture production and are widely used as a model species to gain knowledge of many aspects of fish biology. The common ancestor of the salmonids experienced a whole genome duplication event, making extant salmonids such as the rainbow trout an excellent model for studying the evolution of tetraploidization and re-diploidization in vertebrates. However, the lack of a reference genome sequence hampers research progress for both academic and applied purposes. In order to enrich the genomic tools already available in this species and provide further insight on the complexity of its genome, we sequenced a large number of rainbow trout BAC-end sequences (BES) and characterized their contents.ResultsA total of 176,485 high quality BES, were generated, representing approximately 4% of the trout genome. BES analyses identified 6,848 simple sequence repeats (SSRs), of which 3,854 had high quality flanking sequences for PCR primers design. The first rainbow trout repeat elements database (INRA RT rep1.0) containing 735 putative repeat elements was developed, and identified almost 59.5% of the BES database in base-pairs as repetitive sequence. Approximately 55% of the BES reads (97,846) had more than 100 base pairs of contiguous non-repetitive sequences. The fractions of the 97,846 non-repetitive trout BES reads that had significant BLASTN hits against the zebrafish, medaka and stickleback genome databases were 15%, 16.2% and 17.9%, respectively, while the fractions of the non-repetitive BES reads that had significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 10.7%, 9.5% and 9.5%, respectively. Comparative genomics using paired BAC-ends revealed several regions of conserved synteny across all the fish species analyzed in this study.ConclusionsThe characterization of BES provided insights on the rainbow trout genome. The discovery of specific repeat elements will facilitate analyses of sequence content (e.g. for SNPs discovery and for transcriptome characterization) and future genome sequence assemblies. The numerous microsatellites will facilitate integration of the linkage and physical maps and serve as valuable resource for fine mapping QTL and positional cloning of genes affecting aquaculture production traits. Furthermore, comparative genomics through BES can be used for identifying positional candidate genes from QTL mapping studies, aid in future assembly of a reference genome sequence and elucidating sequence content and complexity in the rainbow trout genome.

Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

BackgroundDNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells.ResultsExamining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines.ConclusionsComparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.

Genomic Selection Identifies Vertebrate Transcription Factor Fezf2 Binding Sites and Target Genes

Identification of transcription factor targets is critical to understanding gene regulatory networks. Here, we uncover transcription factor binding sites and target genes employing systematic evolution of ligands by exponential enrichment (SELEX). Instead of selecting randomly synthesized DNA oligonucleotides as in most SELEX studies, we utilized zebrafish genomic DNA to isolate fragments bound by Fezf2, an evolutionarily conserved gene critical for vertebrate forebrain development. This is, to our knowledge, the first time that SELEX is applied to a vertebrate genome. Computational analysis of bound genomic fragments predicted a core consensus binding site, which identified response elements that mediated Fezf2-dependent transcription both in vitro and in vivo. Fezf2-bound fragments were enriched for conserved sequences. Surprisingly, ∼20% of these fragments overlapped well annotated protein-coding exons. Through loss of function, gain of function, and chromatin immunoprecipitation, we further identified and validated eomesa/tbr2 and lhx2b as biologically relevant target genes of Fezf2. Mutations in eomesa/tbr2 cause microcephaly in humans, whereas lhx2b is a critical regulator of cell fate and axonal targeting in the developing forebrain. These results demonstrate the feasibility of employing genomic SELEX to identify vertebrate transcription factor binding sites and target genes and reveal Fezf2 as a transcription activator and a candidate for evaluation in human microcephaly.

Effect of rad50 mutation on illegitimate recombination in Saccharomyces cerevisiae

Genes in the RAD52 epistasis group are involved in repairing DNA double-stranded breaks via homologous recombination. We have previously shown that RAD50 is involved in mitotic nonhomologous integration but not in homologous integration. However, the role of Rad50 in nonhomologous integration has not previously been examined. In the current work, we report that the rad50∆ mutation caused a tenfold decrease in the frequency of nonhomologous integration with the majority of nonhomologous integrants showing an unstable Ura(+) phenotype. Sequencing analysis of the integration target sites showed that integration events of both ends of the integrating vector in the rad50∆ mutant occurred at different chromosomal locations, resulting in large deletions or translocations on the genomic insertion sites. Interestingly, 47% of events in the rad50∆ mutant were integrated into repetitive sequences including rDNA locus, telomeres and Ty elements and 27% of events were integrated into non-repetitive sequences as compared to 11% of events integrated into rDNA and 70% into non-repetitive sequences in the wild-type cells. These results showed that deletion of RAD50 significantly changes the distribution of different classes of integration events, suggesting that Rad50 is required for nonhomologous integration at non-repetitive sequences more so than at repetitive ones. Furthermore, Southern analysis indicated that half of the events contained deletions at one or at both ends of the integrating DNA fragment, suggesting that Rad50 might have a role in protecting free ends of double-strand breaks. In contrast to the rad50∆ mutant, the rad50S mutant (separation of function allele) slightly increases the frequency of nonhomologous integration but the distribution of integration events is similar to that of wild-type cells with the majority of events integrated into a chromosomal locus. Our results suggest that deletion of RAD50 may block the major pathway of nonhomologous integration into a non-repetitive chromosomal locus and Rad50 may be involved in tethering two ends of the integrating DNA into close proximity that facilitates nonhomologous integration of both ends into a single chromosomal locus.

Improvement of resistance to maize dwarf mosaic virus mediated by transgenic RNA interference

To overcome the low efficiency of agronomic protection from maize dwarf mosaic disease, susceptible maize inbred line was transformed by Agrobacterium tumefaciens harboring hpRNA expression vectors containing inverted-repeat sequences of different lengths targeting coat protein gene ( CP) of maize dwarf mosaic virus (MDMV). After PCR screening and Southern blotting, the flanking sequences of the integration sites were amplified by thermal asymmetric interlaced PCR (TAIL-PCR) and used for analysis of T-DNA integration patterns. The T 2 plant lines were evaluated for their MDMV resistance in field inoculation trials under two environments. Of the nineteen T 2 plant lines positive in Southern blotting, six were evaluated as resistant to MDMV, and four of them had resistance non-significantly different from the highly resistant control “H9-21”, while the resistance of the other eleven was proved to be significantly improved when compared to their non-transformed parent line. These improvements in MDMV resistance were verified by the relative amount of virus CP gene expression measured by quantitative real time PCR. Comparing the results of Southern blotting and TAIL-PCR analysis, different integration patterns of one or two copies of the inverted-repeat sequences were identified from non-repetitive and repetitive sequences of the maize genome. The MDMV resistance mediated by RNA interference is relative to the length of the inverted-repeat sequence, the copy number of T-DNA integration and the repeatability of integration sites. A longer hpRNA expression construct shows more efficiency than a shorter one.

Deep resequencing of serial sputum isolates of Mycobacterium tuberculosis during therapeutic failure due to poor compliance reveals stepwise mutation of key resistance genes on an otherwise stable genetic background

It has generally been held that the repeated emergence of resistance in Mycobacterium tuberculosis is due to the effects of large population sizes, slow replication, and prolonged colonization and treatment. However, there have been suggestions that its emergence is facilitated by high mutation rates due to a lack of mismatch repair, error-prone polymerases, and a potentially mutagenic host niche. Genome re-sequencing has indicated higher variability in strains with emergent resistance, but these studies have not been performedin serial isolates in which drug resistance has emerged. We have used genome re-sequencing to address the mutational processes that occur during the evolution of drug resistance during a clinical infection. Serial isolates from a patient obtained over a 12 month period, and spanning the transition of the colonizing population from fully drug sensitive, to isoniazid resistant, to isoniazid and rifampicin (multiply drug) resistant, spanning an estimated minimum of 100 generations within the host, were deep sequenced using Illumina sequencing. The genomes were compared, and all mutations in non-repetitive sequences were identified. Specific mutations conferring resistance were identified. No additional mutations in non-repetitive regions were present. The mutations observed were kat S315T and rpoB D516Y. M. tuberculosis is relatively stable genetically within the host, and demonstrates greater stability than is suggested by in vitro studies of emergent drug resistance, or by models of hypermutability. This indicates that it is primarily the nature and duration of the infection that are sufficient to lead to the repeated emergence of drug resistance in this infection if improperly managed, and that the selective pressure of the drugs limits additional diversification. This emphasizes the central importance of maintaining therapeutic concentrations of at least two effective antibiotics for the duration of treatment to prevent the emergence of resistance.

Sequence Analysis of SSR-Flanking Regions Identifies Genome Affinities between Pasture Grass Fungal Endophyte Taxa

Fungal species of the Neotyphodium and Epichloë genera are endophytes of pasture grasses showing complex differences of life-cycle and genetic architecture. Simple sequence repeat (SSR) markers have been developed from endophyte-derived expressed sequence tag (EST) collections. Although SSR array size polymorphisms are appropriate for phenetic analysis to distinguish between taxa, the capacity to resolve phylogenetic relationships is limited by both homoplasy and heteroploidy effects. In contrast, nonrepetitive sequence regions that flank SSRs have been effectively implemented in this study to demonstrate a common evolutionary origin of grass fungal endophytes. Consistent patterns of relationships between specific taxa were apparent across multiple target loci, confirming previous studies of genome evolution based on variation of individual genes. Evidence was obtained for the definition of endophyte taxa not only through genomic affinities but also by relative gene content. Results were compatible with the current view that some asexual Neotyphodium species arose following interspecific hybridisation between sexual Epichloë ancestors. Phylogenetic analysis of SSR-flanking regions, in combination with the results of previous studies with other EST-derived SSR markers, further permitted characterisation of Neotyphodium isolates that could not be assigned to known taxa on the basis of morphological characteristics.

Highly nonrepetitive sequences: Winning strategies from the local lemma

AbstractWe prove game‐theoretic versions of several classical results on nonrepetitive sequences, showing the existence of winning strategies using an extension of the Lovász Local Lemma which can dramatically reduce the number of edges needed in a dependency graph when there is an ordering underlying the significant dependencies of events. This appears to represent the first successful application of a Local Lemma to games. © 2010 Wiley Periodicals, Inc. Random Struct. Alg., 2011

FEN1 Ensures Telomere Stability by Facilitating Replication Fork Re-initiation

Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.

MER41 Repeat Sequences Contain Inducible STAT1 Binding Sites

Chromatin immunoprecipitation combined with massively parallel sequencing methods (ChIP-seq) is becoming the standard approach to study interactions of transcription factors (TF) with genomic sequences. At the example of public STAT1 ChIP-seq data sets, we present novel approaches for the interpretation of ChIP-seq data.We compare recently developed approaches to determine STAT1 binding sites from ChIP-seq data. Assessing the content of the established consensus sequence for STAT1 binding sites, we find that the usage of “negative control” ChIP-seq data fails to provide substantial advantages. We derive a single refined probabilistic model of STAT1 binding sequences from these ChIP-seq data. Contrary to previous claims, we find no evidence that STAT1 binds to multiple distinct motifs upon interferon-gamma stimulation in vivo. While a large majority of genomic sites with high ChIP-seq signal is associated with a nucleotide sequence ressembling a STAT1 binding site, only a very small subset of the over 5 million potential STAT1 binding sites in the human genome is covered by ChIP-seq data. Furthermore a surprisingly large fraction of the ChIP-seq signal (5%) is absorbed by a small family of repetitive sequences (MER41). The observation of the binding of activated STAT1 protein to a specific repetitive element bolsters similar reports concerning p53 and other TFs, and strengthens the notion of an involvement of repeats in gene regulation. Incidentally MER41 are specific to primates, consequently, regulatory mechanisms in the IFN-STAT pathway might fundamentally differ between primates and rodents. On a methodological aspect, the presence of large numbers of nearly identical binding sites in repetitive sequences may lead to wrong conclusions about intrinsic binding preferences of TF as illustrated by the spacing analysis STAT1 tandem motifs. Therefore, ChIP-seq data should be analyzed independently within repetitive and non-repetitive sequences.