Functional deficiency of the FEN1 gene has been suggested to cause genomic instability and cancer predisposition. We have identified a group of FEN1 mutations in human cancer specimens. Most of these mutations abrogated two of three nuclease activities of flap endonuclease 1 (FEN1). To demonstrate the etiological significance of these somatic mutations, we inbred a mouse line harboring the E160D mutation representing mutations identified in human cancers. Selective elimination of nuclease activities led to frequent spontaneous mutations and accumulation of incompletely digested DNA fragments in apoptotic cells. The mutant mice were predisposed to autoimmunity, chronic inflammation and cancers. The mutator phenotype results in the initiation of cancer, whereas chronic inflammation promotes the cancer progression. The current work exemplifies the approach of studying the mechanisms of individual polymorphisms and somatic mutations in cancer development, and may serve as a reference in developing new therapeutic regimens through the suppression of inflammatory responses.

Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3-extrahelical nucleotide (3-flap) binding pocket. When presented with 5-flap substrates having a 3-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3-flap on substrates reduced K m and increased multiple-and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3-flap, and the absence of a 3-flap from a 5-flap substrate was more detrimental to hFEN1 activity than removal of the 5-flap or introduction of a hairpin into the 5-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5-phosphorylated product. Together the results indicate that the presence of a 3-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.In eukaryotic DNA replication and repair, various bifurcated nucleic acid structure intermediates are formed and must be processed by the appropriate nuclease. Two examples of biological processes that create bifurcated DNA intermediates are Okazaki fragment maturation (1, 2) and long patch excision repair (3). In both models, a polymerase executes strand-displacement synthesis to create a double-stranded DNA (dsDNA) 6 two-way junction from which a 5Ј-flap structure protrudes. The penultimate step of both pathways is the cleavage of this flap structure to create a nicked DNA that is then ligated. Because the bifurcated DNA structures that are formed in the aforementioned processes can theoretically occur anywhere in the genome, the nuclease associated with the cleavage of 5Ј-flap structures in eukaryotic cells, which is called flap endonuclease 1 (FEN1), must be capable of cleavage regardless of sequence. Therefore, FEN1 nucleases, which are found in all kingdoms of life (4), have evolved to recognize substrates based upon nucleic acid structure and strand polarity (5, 6).The Okazaki fragment maturation pathway of yeast has become a paradigm of eukaryotic lagging strand DNA synthesis. In the yeast model, bifurcated intermediates with large single-stranded DNA (ssDNA) 5Ј-flap structures are imprecisely cleaved by DNA2 ...

Flap EndoNuclease-1 (FEN-1) is a multifunctional and structure-specific nuclease involved in nucleic acid processing pathways. It plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair and resolution of dinucleotide and trinucleotide repeat secondary structures. In addition to its flap endonuclease (FEN) and nick exonuclease (EXO) activities, a new gap endonuclease (GEN) activity has been characterized. This activity may be important in apoptotic DNA fragmentation and in resolving stalled DNA replication forks. The multiple functions of FEN-1 are regulated via several means, including formation of complexes with different protein partners, nuclear localization in response to cell cycle or DNA damage and post-translational modifications. Its functional deficiency is predicted to cause genetic diseases, including Huntington's disease, myotonic dystrophy and cancers. This review summarizes the knowledge gained through efforts in the past decade to define its structural elements for specific activities and possible pathological consequences of altered functions of this multirole player.

BackgroundExposure to environmental endocrine-disrupting chemicals during pregnancy reportedly causes transgenerationally inherited reproductive defects. We hypothesized that to affect the grandchild, endocrine-disrupting chemicals must alter the epigenome of the germ cells of the in utero-exposed G1 male fetus. Additionally, to affect the great-grandchild, the aberration must persist in the germ cells of the unexposed G2 grandchild.ResultsHere, we treat gestating female mice with vinclozolin, bisphenol A, or di-(2-ethylhexyl)phthalate during the time when global de novo DNA methylation and imprint establishment occurs in the germ cells of the G1 male fetus. We map genome-wide features in purified G1 and G2 prospermatogonia, in order to detect immediate and persistent epigenetic aberrations, respectively. We detect changes in transcription and methylation in the G1 germline immediately after endocrine-disrupting chemicals exposure, but changes do not persist into the G2 germline. Additional analysis of genomic imprints shows no persistent aberrations in DNA methylation at the differentially methylated regions of imprinted genes between the G1 and G2 prospermatogonia, or in the allele-specific transcription of imprinted genes between the G2 and G3 soma.ConclusionsOur results suggest that endocrine-disrupting chemicals exert direct epigenetic effects in exposed fetal germ cells, which are corrected by reprogramming events in the next generation. Avoiding transgenerational inheritance of environmentally-caused epigenetic aberrations may have played an evolutionary role in the development of dual waves of global epigenome reprogramming in mammals.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0619-z) contains supplementary material, which is available to authorized users.

Very few studies have addressed the diversity of culturable fungi from deep-sea sediments. We are reporting here the diversity of culturable fungi in deep-sea sediments of the Central Indian Basin obtained from a depth of ∼5,000 m. A total of 16 filamentous fungi and 12 yeasts isolated from 20 sediment cores of ∼35 cm length were identified by 18S and ITS sequencing of SSU rDNA. Most of the filamentous fungi were Ascomycota, while two were Basidiomycota. Microscopic identification of sporulating cultures mostly matched either with 18S or ITS sequences but seldom with both the sequences. Phylogenetic analysis of ascomycetes using 18S sequence data grouped them into 7 clusters belonging to Aspergillus sp., Sagenomella sp., Exophiala sp., Capronia sp., Cladosporium sp., Acremonium sp. and Tritirachium sp. ITS sequence data grouped isolates into 6 clusters belonging to Aspergillus sp., uncultured member of Hypocreaceae, Exophiala sp., uncultured soil fungus, Hypocreales and Trichothecium sp. The two basidiomycete isolates were a Tilletiopsis sp. evident from 18S as well as ITS sequence data. In contrast, most of the yeast isolates belonged to Basidiomycota and only one isolate belonged to the phylum Ascomycota. Sequences of 18S as well as ITS gave matching identification of most of the yeasts. Filamentous fungi as well as the yeasts grew at 200 bar/5°C indicating their adaptations to deep-sea conditions. This is the first report on isolation of Sagenomella, Exophiala, Capronia and Tilletiopsis spp. from deep-sea sediments. This study reports on the presence of terrestrial fungi as a component of culturable fungi in deep-sea sediments.

We report a modifying effect of a polymorphism of CELF4 (rs1786814) on the dose-dependent association between anthracyclines and cardiomyopathy, which possibly occurs through a pathway that involves the expression of abnormally spliced TNNT2 variants.

SUMMARY To understand what dictates the emerging patterns of de novo DNA methylation in the male germline, we mapped DNA methylation, chromatin, and transcription changes in purified fetal mouse germ cells by using methylated CpG island recovery assay (MIRA)-chip, chromatin immunoprecipitation (ChIP)-chip, and strand-specific RNA deep sequencing, respectively. Global de novo methylation occurred by default in prospermatogonia without any apparent trigger from preexisting repressive chromatin marks but was preceded by broad, low-level transcription along the chromosomes, including the four known paternally imprinted differentially methylated regions (DMRs). Default methylation was excluded only at precisely aligned constitutive or emerging peaks of H3K4me2, including most CpG islands and some intracisternal A particles (IAPs). Similarly, each maternally imprinted DMR was protected from default DNA methylation among highly methylated DNA by an H3K4me2 peak and transcription initiation at least in one strand. Our results suggest that the pattern of de novo DNA methylation in prospermatogonia is dictated by opposing actions of broad, low-level transcription and dynamic patterns of active chromatin.

Imprinted gene expression corresponds to parental allele-specific DNA CpG methylation and chromatin composition. Histone tail covalent modifications have been extensively studied, but it is not known whether modifications in the histone globular domains can also discriminate between the parental alleles. Using multiplex chromatin immunoprecipitation-single nucleotide primer extension (ChIP-SNuPE) assays, we measured the allele-specific enrichment of H3K79 methylation and H4K91 acetylation along the H19/Igf2 imprinted domain. Whereas H3K79me1, H3K79me2, and H4K91ac displayed a paternal-specific enrichment at the paternally expressed Igf2 locus, H3K79me3 was paternally biased at the maternally expressed H19 locus, including the paternally methylated imprinting control region (ICR). We found that these allele-specific differences depended on CTCF binding in the maternal ICR allele. We analyzed an additional 11 differentially methylated regions (DMRs) and found that, in general, H3K79me3 was associated with the CpG-methylated alleles, whereas H3K79me1, H3K79me2, and H4K91ac enrichment was specific to the unmethylated alleles. Our data suggest that allele-specific differences in the globular histone domains may constitute a layer of the "histone code" at imprinted genes.