Electronic control of polymerase binding to DNA

Electronic Control of DNA Polymerase Binding and Unbinding to Single DNA Molecules.
Noah A. Wilson, Robin Abu-Shumays, Brett Gyarfas, Hongyun Wang, Kate R. Lieberman, Mark Akeson, William B. Dunbar.
ACS Nano, Article ASAP | DOI:10.1021/nn9000897

DNA polymerases catalyze template-dependent genome replication. The assembly of a high affinity ternary complex between these enzymes, the double strand－single strand junction of their DNA substrate, and the deoxynucleoside triphosphate (dNTP) complementary to the first template base in the polymerase active site is essential to this process. We present a single molecule method for iterative measurements of DNA－polymerase complex assembly with high temporal resolution, using active voltage control of individual DNA substrate molecules tethered noncovalently in an α-hemolysin nanopore. DNA binding states of the Klenow fragment of Escherichia coli DNA polymerase I (KF) were diagnosed based upon their ionic current signature, and reacted to with submillisecond precision to execute voltage changes that controlled exposure of the DNA substrate to KF and dNTP. Precise control of exposure times allowed measurements of DNA－KF complex assembly on a time scale that superimposed with the rate of KF binding. Hundreds of measurements were made with a single tethered DNA molecule within seconds, and dozens of molecules can be tethered within a single experiment. This approach allows statistically robust analysis of the assembly of complexes between DNA and RNA processing enzymes and their substrates at the single molecule level.

# せっかくKUくんに教えてもらっていたのに忘れてた…すまそ＞KU君

Slowing down DNA in nanopore sequencing

Reverse DNA translocation through a solid-state nanopore by magnetic tweezers.
Hongbo Peng, Xinsheng Sean Ling.
Nanotechnology 20, 185101 (2009) | doi:10.1088/0957-4484/20/18/185101

Voltage-driven DNA translocation through nanopores has attracted wide interest for many potential applications in molecular biology and biotechnology. However, it is intrinsically difficult to control the DNA motion in standard DNA translocation processes in which a strong electric field is required in drawing DNA into the pore, but it also leads to uncontrollable fast DNA translocation. Here we explore a new type of DNA translocation. We dub it 'reverse DNA translocation', in which the DNA is pulled through a nanopore mechanically by a magnetic bead, driven by a magnetic-field gradient. This technique is compatible with simultaneous ionic current measurements and is suitable for multiple nanopores, paving the way for large scale applications. We report the first experiment of reverse DNA translocation through a solid-state nanopore using magnetic tweezers.

# Nanoporeを通るDNAがあまりにも速くて読み取れない上にコントロール不能なので、足かせして2,000倍以上遅くしたそうな…

幻影4

The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line.
The FANTOM Consortium, Riken Omics Science Center.
Nature Genetics, Advance online publication | doi:10.1038/ng.375 | PMID:

Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process.

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The regulated retrotransposon transcriptome of mammalian cells.
Geoffrey J Faulkner, Yasumasa Kimura, Carsten O Daub, Shivangi Wani, Charles Plessy, Katharine M Irvine, Kate Schroder, Nicole Cloonan, Anita L Steptoe, Timo Lassmann, Kazunori Waki, Nadine Hornig, Takahiro Arakawa, Hazuki Takahashi, Jun Kawai, Alistair R R Forrest, Harukazu Suzuki, Yoshihide Hayashizaki, David A Hume, Valerio Orlando, Sean M Grimmond, Piero Carninci.
Nature Genetics, Advance online publication | doi:10.1038/ng.368 | PMID:

Although repetitive elements pervade mammalian genomes, their overall contribution to transcriptional activity is poorly defined. Here, as part of the FANTOM4 project, we report that 6–30% of cap-selected mouse and human RNA transcripts initiate within repetitive elements. Analysis of approximately 250,000 retrotransposon-derived transcription start sites shows that the associated transcripts are generally tissue specific, coincide with gene-dense regions and form pronounced clusters when aligned to full-length retrotransposon sequences. Retrotransposons located immediately 5' of protein-coding loci frequently function as alternative promoters and/or express noncoding RNAs. More than a quarter of RefSeqs possess a retrotransposon in their 3' UTR, with strong evidence for the reduced expression of these transcripts relative to retrotransposon-free transcripts. Finally, a genome-wide screen identifies 23,000 candidate regulatory regions derived from retrotransposons, in addition to more than 2,000 examples of bidirectional transcription. We conclude that retrotransposon transcription has a key influence upon the transcriptional output of the mammalian genome.

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Tiny RNAs associated with transcription start sites in animals.
Ryan J Taft, Evgeny A Glazov, Nicole Cloonan, Cas Simons, Stuart Stephen, Geoffrey J Faulkner, Timo Lassmann, Alistair R R Forrest, Sean M Grimmond, Kate Schroder, Katharine Irvine, Takahiro Arakawa, Mari Nakamura, Atsutaka Kubosaki, Kengo Hayashida, Chika Kawazu, Mitsuyoshi Murata, Hiromi Nishiyori, Shiro Fukuda, Jun Kawai, Carsten O Daub, David A Hume, Harukazu Suzuki, Valerio Orlando, Piero Carninci, Yoshihide Hayashizaki, John S Mattick.
Nature Genetics, Advance online publication | doi:10.1038/ng.312 | PMID:

It has been reported that relatively short RNAs of heterogeneous sizes are derived from sequences near the promoters of eukaryotic genes. In conjunction with the FANTOM4 project, we have identified tiny RNAs with a modal length of 18 nt that map within -60 to +120 nt of transcription start sites (TSSs) in human, chicken and Drosophila. These transcription initiation RNAs (tiRNAs) are derived from sequences on the same strand as the TSS and are preferentially associated with G+C-rich promoters. The 5' ends of tiRNAs show peak density 10–30 nt downstream of TSSs, indicating that they are processed. tiRNAs are generally, although not exclusively, associated with highly expressed transcripts and sites of RNA polymerase II binding. We suggest that tiRNAs may be a general feature of transcription in metazoa and possibly all eukaryotes.

# 一応…

Sixth Nucleotide

The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain.
Skirmantas Kriaucionis, Nathaniel Heintz.
Science, Science Express | DOI:10.1126/science.1169786 | PMID:

In spite of the importance of epigenetic regulation in neurological disorders, little is known about neuronal chromatin. Cerebellar Purkinje neurons have large and euchromatic nuclei, whilst granule cell nuclei are small and have a more typical heterochromatin distribution. While comparing the abundance of 5-methylcytosine (mC) in Purkinje and granule cell nuclei, we detected the presence of an unusual DNA nucleotide. Using thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), and mass spectrometry (MS), we have identified the nucleotide as 5-hydroxymethyl-2’-deoxycytidine (hmdC). hmdC comprises 0.6% of total nucleotides in Purkinje cells, 0.2% in granule cells, and is not present in cancer cell lines. hmdC is a constituent of nuclear DNA that is enriched in the brain, suggesting a role in epigenetic control of neuronal function.

メチル化Cだけでなくヒドロキシメチル化Cもエピジェネティカルな働きを持っているそうな。これは要チェックでしょう。

Nano litmus made from DNA

A DNA nanomachine that maps spatial and temporal pH changes inside living cells.
Souvik Modi, Swetha M. G., Debanjan Goswami, Gagan D. Gupta, Satyajit Mayor, Yamuna Krishnan.
Nature Nanotechnology, Advance online publication | doi:10.1038/nnano.2009.83 | PMID:

DNA nanomachines are synthetic assemblies that switch between defined molecular conformations upon stimulation by external triggers. Previously, the performance of DNA devices has been limited to in vitro applications. Here we report the construction of a DNA nanomachine called the I-switch, which is triggered by protons and functions as a pH sensor based on fluorescence resonance energy transfer (FRET) inside living cells. It is an efficient reporter of pH from pH 5.5 to 6.8, with a high dynamic range between pH 5.8 and 7. To demonstrate its ability to function inside living cells we use the I-switch to map spatial and temporal pH changes associated with endosome maturation. The performance of our DNA nanodevices inside living systems illustrates the potential of DNA scaffolds responsive to more complex triggers in sensing, diagnostics and targeted therapies in living systems.

細胞内微小局所のpHをpH5.8～7の間で検知可能…それよりも、ナノツールを細胞内に見事に運搬しているところを見習わねば…。