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Nucleic Acids Research: VOLUME 37 ISSUE 20 2009

Fri, 13 Nov 2009 06:41:12 PST

Nucleic Acids Research

Fri, 13 Nov 2009 06:41:12 PST

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Fri, 13 Nov 2009 06:41:12 PST

Real-time DNA microarray analysis

Hassibi, A., Vikalo, H., Riechmann, J. L., Hassibi, B. — Fri, 13 Nov 2009 06:41:14 PST

We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e. real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation in the capturing spots, washing artifacts, microarray spot-to-spot variations, and other signal amplitude-affecting non-idealities. We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays.

In vivo expression and purification of aptamer-tagged small RNA regulators

Said, N., Rieder, R., Hurwitz, R., Deckert, J., Urlaub, H., Vogel, J. — Fri, 13 Nov 2009 06:41:14 PST

Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA–protein complexes in a wide range of bacteria.

Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production

Fri, 13 Nov 2009 06:41:14 PST

In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines.

A generalized conformational energy function of DNA derived from molecular dynamics simulations

Yamasaki, S., Terada, T., Shimizu, K., Kono, H., Sarai, A. — Fri, 13 Nov 2009 06:41:14 PST

Proteins recognize DNA sequences by two different mechanisms. The first is direct readout, in which recognition is mediated by direct interactions between the protein and the DNA bases. The second is indirect readout, which is caused by the dependence of conformation and the deformability of the DNA structure on the sequence. Various energy functions have been proposed to evaluate the contribution of indirect readout to the free-energy changes in complex formations. We developed a new generalized energy function to estimate the dependence of the deformability of DNA on the sequence. This function was derived from molecular dynamics simulations previously conducted on B-DNA dodecamers, each of which had one possible tetramer sequence embedded at its center. By taking the logarithm of the probability distribution function (PDF) for the base-step parameters of the central base-pair step of the tetramer, its ability to distinguish the native sequence from random ones was superior to that with the previous method that approximated the energy function in harmonic form. From a comparison of the energy profiles calculated with these two methods, we found that the harmonic approximation caused significant errors in the conformational energies of the tetramers that adopted multiple stable conformations.

A high-resolution magnetic tweezer for single-molecule measurements

Kim, K., Saleh, O. A. — Fri, 13 Nov 2009 06:41:14 PST

Magnetic tweezers (MT) are single-molecule manipulation instruments that utilize a magnetic field to apply force to a biomolecule-tethered magnetic bead while using optical bead tracking to measure the biomolecule’s extension. While relatively simple to set up, prior MT implementations have lacked the resolution necessary to observe sub-nanometer biomolecular configuration changes. Here, we demonstrate a reflection-interference technique for bead tracking, and show that it has much better resolution than traditional diffraction-based systems. We enhance the resolution by fabricating optical coatings on all reflecting surfaces that optimize the intensity and contrast of the interference image, and we implement feedback control of the focal position to remove drift. To test the system, we measure the length change of a DNA hairpin as it undergoes a folding/unfolding transition.

A high throughput experimental approach to identify miRNA targets in human cells

Fri, 13 Nov 2009 06:41:14 PST

The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation—gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that ~40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.

FIGfams: yet another set of protein families

Meyer, F., Overbeek, R., Rodriguez, A. — Fri, 13 Nov 2009 06:41:12 PST

We present FIGfams, a new collection of over 100 000 protein families that are the product of manual curation and close strain comparison. Using the Subsystem approach the manual curation is carried out, ensuring a previously unattained degree of throughput and consistency. FIGfams are based on over 950 000 manually annotated proteins and across many hundred Bacteria and Archaea. Associated with each FIGfam is a two-tiered, rapid, accurate decision procedure to determine family membership for new proteins. FIGfams are freely available under an open source license. These can be downloaded at ftp://ftp.theseed.org/FIGfams/. The web site for FIGfams is http://www.theseed.org/wiki/FIGfams/

Transcription from bacteriophage {lambda} pR promoter is regulated independently and antagonistically by DksA and ppGpp

Lyzen, R., Kochanowska, M., Wegrzyn, G., Szalewska-Palasz, A. — Fri, 13 Nov 2009 06:41:12 PST

The stringent response effector, guanosine tetraphosphate (ppGpp), adjust gene expression and physiology in bacteria, by affecting the activity of various promoters. RNA polymerase-interacting protein, DksA, was proposed to be the co-factor of ppGpp effects; however, there are reports suggesting independent roles of these regulators. Bacteriophage major lytic promoter, pR, is down-regulated by the stringent response and ppGpp. Here, we present evidence that DksA significantly stimulates pR-initiated transcription in vitro in the reconstituted system. DksA is also indispensable for pR activity in vivo. DksA-mediated activation of pR-initiated transcription is predominant over ppGpp effects in the presence of both regulators in vitro. The possible role of the opposite regulation by ppGpp and DksA in phage development is discussed. The major mechanism of DksA-mediated activation of transcription from pR involves facilitating of RNA polymerase binding to the promoter region, which results in more productive transcription initiation. Thus, our results provide evidence for the first promoter inhibited by ppGpp that can be stimulated by the DksA protein both in vivo and in vitro. Therefore, DksA role could be not only independent but antagonistic to ppGpp in transcription regulation.

The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

Fri, 13 Nov 2009 06:41:12 PST

In Drosophila, the dADA2b-containing dSAGA complex is involved in histone H3 lysine 9 and 14 acetylation. Curiously, although the lysine 9- and 14-acetylated histone H3 levels are drastically reduced in dAda2b mutants, these animals survive until a late developmental stage. To study the molecular consequences of the loss of histone H3 lysine 9 and 14 acetylation, we compared the total messenger ribonucleic acid (mRNA) profiles of wild type and dAda2b mutant animals at two developmental stages. Global gene expression profiling indicates that the loss of dSAGA-specific H3 lysine 9 and 14 acetylation results in the expression change (up- or down-regulation) of a rather small subset of genes and does not cause a general transcription de-regulation. Among the genes up-regulated in dAda2b mutants, particularly high numbers are those which play roles in antimicrobial defense mechanisms. Results of chromatin immunoprecipitation experiments indicate that in dAda2b mutants, the lysine 9-acetylated histone H3 levels are decreased both at dSAGA up- and down-regulated genes. In contrast to that, in the promoters of dSAGA-independent ribosomal protein genes a high level of histone H3K9ac is maintained in dAda2b mutants. Our data suggest that by acetylating H3 at lysine 9, dSAGA modifies Pol II accessibility to specific promoters differently.

MeCP2/H3meK9 are involved in IL-6 gene silencing in pancreatic adenocarcinoma cell lines

Dandrea, M., Donadelli, M., Costanzo, C., Scarpa, A., Palmieri, M. — Fri, 13 Nov 2009 06:41:12 PST

The aim of the present study was to analyse the molecular mechanisms involved in the Interleukin-6 (IL-6) silencing in pancreatic adenocarcinoma cell lines. Our results demonstrate that TNF-, a major IL-6 inducer, is able to induce IL-6 only in three out of six cell lines examined. 5-aza-2'-deoxycytidine (DAC), but not trichostatin A (TSA), activates the expression of IL-6 in all cell lines, indicating that DNA methylation, but not histone deacetylation, plays an essential role in IL-6 silencing. Indeed, the IL-6 upstream region shows a methylation status that correlates with IL-6 expression and binds MeCP2 and H3meK9 only in the non-expressing cell lines. Our results suggest that critical methylations located from positions –666 to –426 relative to the transcription start site of IL-6 may act as binding sites for MeCP2.

GSK3{beta} is a negative regulator of the transcriptional coactivator MAML1

Fri, 13 Nov 2009 06:41:12 PST

Glycogen synthase kinase 3β (GSK3β) is involved in several cellular signaling systems through regulation of the activity of diverse transcription factors such as Notch, p53 and β-catenin. Mastermind-like 1 (MAML1) was originally identified as a Notch coactivator, but has also been reported to function as a transcriptional coregulator of p53, β-catenin and MEF2C. In this report, we show that active GSK3β directly interacts with the MAML1 N-terminus and decreases MAML1 transcriptional activity, suggesting that GSK3β might target a coactivator in its regulation of gene expression. We have previously shown that MAML1 increases global acetylation of histones, and here we show that the GSK3 inhibitor SB41, further enhances MAML1-dependent histone acetylation in cells. Finally, MAML1 translocates GSK3β to nuclear bodies; this function requires full-length MAML1 protein.

In vivo methylation of mtDNA reveals the dynamics of protein-mtDNA interactions

Rebelo, A. P., Williams, S. L., Moraes, C. T. — Fri, 13 Nov 2009 06:41:12 PST

To characterize the organization of mtDNA–protein complexes (known as nucleoids) in vivo, we have probed the mtDNA surface exposure using site-specific DNA methyltransferases targeted to the mitochondria. We have observed that DNA methyltransferases have different accessibility to different sites on the mtDNA based on the levels of protein occupancy. We focused our studies on selected regions of mtDNA that are believed to be major regulatory regions involved in transcription and replication. The transcription termination region (TERM) within the tRNA^Leu(UUR) gene was consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mitochondrial transcription termination factor 1 (mTERF1) to the site. Protection from methylation was also observed in other regions of the mtDNA, including the light and heavy strand promoters (LSP, HSP) and the origin of replication of the light strand (OL). Manipulations aiming at increasing or decreasing the levels of the mitochondrial transcription factor A (TFAM) led to decreased in vivo methylation, whereas manipulations that stimulated mtDNA replication led to increased methylation. We also analyzed the effect of ATAD3 and oxidative stress in mtDNA exposure. Our data provide a map of human mtDNA accessibility and demonstrate that nucleoids are dynamically associated with proteins.

Genome-wide analysis of a G-quadruplex-specific single-chain antibody that regulates gene expression

Fri, 13 Nov 2009 06:41:12 PST

G-quadruplex nucleic acids have been proposed to play a role in a number of fundamental biological processes that include transcription and translation. We have developed a single-chain antibody that is selective for G-quadruplex DNA over double-stranded DNA, and here show that when it is expressed in human cells, it significantly affects the expression of a wide variety of genes, in a manner that correlates with the presence of predicted G-quadruplexes. We observe cases where gene expression is increased or decreased, and that there are apparent interactions with G-quadruplex motifs at the beginning and end of the genes, and on either strand. The outcomes of this genome-wide study demonstrate that G-quadruplex recognition by the antibody has physiological consequences, and provides insights into some of the complexity associated with G-quadruplex-based regulation.

Regulation of transcription termination in the nematode Caenorhabditis elegans

Fri, 13 Nov 2009 06:41:12 PST

The current predicted mechanisms that describe RNA polymerase II (pol II) transcription termination downstream of protein expressing genes fail to adequately explain, how premature termination is prevented in eukaryotes that possess operon-like structures. Here we address this issue by analysing transcription termination at the end of single protein expressing genes and genes located within operons in the nematode Caenorhabditis elegans. By using a combination of RT-PCR and ChIP analysis we found that pol II generally transcribes up to 1 kb past the poly(A) sites into the 3' flanking regions of the nematode genes before it terminates. We also show that pol II does not terminate after transcription of internal poly(A) sites in operons. We provide experimental evidence that five randomly chosen C. elegans operons are transcribed as polycistronic pre-mRNAs. Furthermore, we show that cis-splicing of the first intron located in downstream positioned genes in these polycistronic pre-mRNAs is critical for their expression and may play a role in preventing premature pol II transcription termination.

Translesion DNA synthesis-assisted non-homologous end-joining of complex double-strand breaks prevents loss of DNA sequences in mammalian cells

Covo, S., de Villartay, J.-P., Jeggo, P. A., Livneh, Z. — Fri, 13 Nov 2009 06:41:12 PST

Double strand breaks (DSB) are severe DNA lesions, and if not properly repaired, may lead to cell death or cancer. While there is considerable data on the repair of simple DSB (sDSB) by non-homologous end-joining (NHEJ), little is known about the repair of complex DSBs (cDSB), namely breaks with a nearby modification, which precludes ligation without prior processing. To study the mechanism of cDSB repair we developed a plasmid-based shuttle assay for the repair of a defined site-specific cDSB in cultured mammalian cells. Using this assay we found that repair efficiency and accuracy of a cDSB with an abasic site in a 5' overhang was reduced compared with a sDSB. Translesion DNA synthesis (TLS) across the abasic site located at the break prevented loss of DNA sequences, but was highly mutagenic also at the template base next to the abasic site. Similar to sDSB repair, cDSB repair was totally dependent on XrccIV, and altered in the absence of Ku80. In contrast, Artemis appears to be specifically involved in cDSB repair. These results may indicate that mammalian cells have a damage control strategy, whereby severe deletions are prevented at the expense of the less deleterious point mutations during NHEJ.

Involvement of p54(nrb), a PSF partner protein, in DNA double-strand break repair and radioresistance

Li, S., Kuhne, W. W., Kulharya, A., Hudson, F. Z., Ha, K., Cao, Z., Dynan, W. S. — Fri, 13 Nov 2009 06:41:12 PST

Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a -H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF·p54(nrb) complex may thus be a potential target for radiosensitizer development.

Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption

Fri, 13 Nov 2009 06:41:12 PST

The SRS2 (Suppressor of RAD Six screen mutant 2) gene encodes an ATP-dependent DNA helicase that regulates homologous recombination in Saccharomyces cerevisiae. Mutations in SRS2 result in a hyper-recombination phenotype, sensitivity to DNA damaging agents and synthetic lethality with mutations that affect DNA metabolism. Several of these phenotypes can be suppressed by inactivating genes of the RAD52 epistasis group that promote homologous recombination, implicating inappropriate recombination as the underlying cause of the mutant phenotype. Consistent with the genetic data, purified Srs2 strongly inhibits Rad51-mediated recombination reactions by disrupting the Rad51-ssDNA presynaptic filament. Srs2 interacts with Rad51 in the yeast two-hybrid assay and also in vitro. To investigate the functional relevance of the Srs2-Rad51 complex, we have generated srs2 truncation mutants that retain full ATPase and helicase activities, but differ in their ability to interact with Rad51. Importantly, the srs2 mutant proteins attenuated for Rad51 interaction are much less capable of Rad51 presynaptic filament disruption. An internal deletion in Srs2 likewise diminishes Rad51 interaction and anti-recombinase activity. We also present evidence that deleting the Srs2 C-terminus engenders a hyper-recombination phenotype. These results highlight the importance of Rad51 interaction in the anti-recombinase function of Srs2, and provide evidence that this Srs2 function can be uncoupled from its helicase activity.

Biophysical characterizations of human mitochondrial transcription factor A and its binding to tumor suppressor p53

Fri, 13 Nov 2009 06:41:12 PST

Human mitochondrial transcription factor A (TFAM) is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a K_d of 1.95 ± 0.19 µM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM–p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices 1 and 2 of the HMG-box constitute the main p53-binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM–p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM–p53 interaction are discussed.

Genome-wide colonization of gene regulatory elements by G4 DNA motifs

Du, Z., Zhao, Y., Li, N. — Fri, 13 Nov 2009 06:41:12 PST

G-quadruplex (or G4 DNA), a stable four-stranded structure found in guanine-rich regions, is implicated in the transcriptional regulation of genes involved in growth and development. Previous studies on the role of G4 DNA in gene regulation mostly focused on genomic regions proximal to transcription start sites (TSSs). To gain a more comprehensive understanding of the regulatory role of G4 DNA, we examined the landscape of potential G4 DNA (PG4Ms) motifs in the human genome and found that G4 motifs, not restricted to those found in the TSS-proximal regions, are bias toward gene-associated regions. Significantly, analyses of G4 motifs in seven types of well-known gene regulatory elements revealed a constitutive enrichment pattern and the clusters of G4 motifs tend to be colocalized with regulatory elements. Considering our analysis from a genome evolutionary perspective, we found evidence that the occurrence and accumulation of certain progenitors and canonical G4 DNA motifs within regulatory regions were progressively favored by natural selection. Our results suggest that G4 DNA motifs are ‘colonized’ in regulatory regions, supporting a likely genome-wide role of G4 DNA in gene regulation. We hypothesize that G4 DNA is a regulatory apparatus situated in regulatory elements, acting as a molecular switch that can modulate the role of the host functional regions, by transition in DNA structure.

Selection for minimization of translational frameshifting errors as a factor in the evolution of codon usage

Huang, Y., Koonin, E. V., Lipman, D. J., Przytycka, T. M. — Fri, 13 Nov 2009 06:41:12 PST

In a wide range of genomes, it was observed that the usage of synonymous codons is biased toward specific codons and codon patterns. Factors that are implicated in the selection for codon usage include facilitation of fast and accurate translation. There are two types of translational errors: missense errors and processivity errors. There is considerable evidence in support of the hypothesis that codon usage is optimized to minimize missense errors. In contrast, little is known about the relationship between codon usage and frameshifting errors, an important form of processivity errors, which appear to occur at frequencies comparable to the frequencies of missense errors. Based on the recently proposed pause-and-slip model of frameshifting, we developed Frameshifting Robustness Score (FRS). We used this measure to test if the pattern of codon usage indicates optimization against frameshifting errors. We found that the FRS values of protein-coding sequences from four analyzed genomes (the bacteria Bacillus subtilis and Escherichia coli, and the yeasts Saccharomyces cerevisiae and Schizosaccharomyce pombe) were typically higher than expected by chance. Other properties of FRS patterns observed in B. subtilis, S. cerevisiae and S. pombe, such as the tendency of FRS to increase from the 5'- to 3'-end of protein-coding sequences, were also consistent with the hypothesis of optimization against frameshifting errors in translation. For E. coli, the results of different tests were less consistent, suggestive of a much weaker optimization, if any. Collectively, the results fit the concept of selection against mistranslation-induced protein misfolding being one of the factors shaping the evolution of both coding and non-coding sequences.

Predictable suppression of gene expression by 5'-UTR-based RNA quadruplexes

Halder, K., Wieland, M., Hartig, J. S. — Fri, 13 Nov 2009 06:41:12 PST

Four-stranded DNA and RNA quadruplexes or G4 motifs are non-B DNA conformations that are presumed to form in vivo, although only few explicit evidence has been reported. Using bioinformatics the presence of putative DNA G-quadruplexes within critical promoter regions has been demonstrated and a regulatory role in transcription has been suspected. However, in genomic DNA the presence of the complementary strand interferes with the potential to form a quadruplex motif. Contrarily RNA G4 motifs have no such limitation and consequently strong interference with gene expression is suspected. Nevertheless, experimental evidence is scarce. Here we show a well-defined structure–function relationship of synthetic quadruplex sequences in 5'-UTRs in multiple mammalian cell-lines. We establish a universal ‘translational suppressor’ effect of these motifs on gene expression at the translational level and show for the first time that specific features such as loop-length and the number of ‘GGG’-repeats further determine the suppressive impact. Moreover, a consistent and predictable repression of gene expression is observed for naturally occurring RNA G4 motifs, augmenting the functional relevance of these unusual nucleic acid structures.

Characterization of RNA aptamers that disrupt the RUNX1-CBF{beta}/DNA complex

Fri, 13 Nov 2009 06:41:13 PST

The transcription factor RUNX1 (AML1) is an important regulator of haematopoiesis, and an important fusion partner in leukaemic translocations. High-affinity DNA binding by RUNX1 requires the interaction of the RUNX1 Runt-Homology-Domain (RHD) with the core-binding factor β protein (CBFβ). To generate novel reagents for in vitro and in vivo studies of RUNX1 function, we have selected high-affinity RNA aptamers against a recombinant RHD–CBFβ complex. Selection yielded two sequence families, each dominated by a single consensus sequence. Aptamers from each family disrupt DNA binding by the RUNX1 protein in vitro and compete with sequence-specific dsDNA binding. Minimal, high-affinity (~100–160 nM) active aptamer fragments 28 and 30 nts in length, consisting of simple short stem-loop structures, were then identified. These bind to the RHD subunit and disrupt its interaction with CBFβ, which is consistent with reduced DNA affinity in the presence of aptamer. These aptamers represent new reagents that target a novel surface on the RHD required to stabilize the recombinant RHD–CBFβ complex and thus will further aid exploring the functions of this key transcription factor.

DNA translocation activity of the multifunctional replication protein ORF904 from the archaeal plasmid pRN1

Sanchez, M., Drechsler, M., Stark, H., Lipps, G. — Fri, 13 Nov 2009 06:41:13 PST

The replication protein ORF904 from the plasmid pRN1 is a multifunctional enzyme with ATPase-, primase- and DNA polymerase activity. Sequence analysis suggests the presence of at least two conserved domains: an N-terminal prim/pol domain with primase and DNA polymerase activities and a C-terminal superfamily 3 helicase domain with a strong double-stranded DNA dependant ATPase activity. The exact molecular function of the helicase domain in the process of plasmid replication remains unclear. Potentially this motor protein is involved in duplex remodelling and/or origin opening at the plasmid replication origin. In support of this we found that the monomeric replication protein ORF904 forms a hexameric ring in the presence of DNA. It is able to translocate along single-stranded DNA in 3'–5' direction as well as on double-stranded DNA. Critical residues important for ATPase activity and DNA translocation activity were identified and are in agreement with a homology model of the helicase domain. In addition we propose that a winged helix DNA-binding domain at the C-terminus of the helicase domain could assist the binding of the replication protein specifically to the replication origin.

Evidence of the crucial role of the linker domain on the catalytic activity of human topoisomerase I by experimental and simulative characterization of the Lys681Ala mutant

Fri, 13 Nov 2009 06:41:13 PST

The functional and structural-dynamical properties of the Lys681Ala mutation in the human topoisomerase IB linker domain have been investigated by catalytic assays and molecular dynamics simulation. The mutant is characterized by a comparable cleavage and a strongly reduced religation rate when compared to the wild type protein. The mutant also displays perturbed linker dynamics, as shown by analysis of the principal components of the motion, and a reduced electrostatic interaction with DNA. Inspection of the inter atomic distances in proximity of the active site shows that in the mutant the distance between the amino group of Lys532 side chain and the 5' OH of the scissile phosphate is longer than the wild type enzyme, providing an atomic explanation for the reduced religation rate of the mutant. Taken together these results indicate the existence of a long range communication between the linker domain and the active site region and points out the crucial role of the linker in the modulation of the catalytic activity.

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Neely, R. K., Tamulaitis, G., Chen, K., Kubala, M., Siksnys, V., Jones, A. C. — Fri, 13 Nov 2009 06:41:13 PST

Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display

Fri, 13 Nov 2009 06:41:13 PST

Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions.

Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny

Fri, 13 Nov 2009 06:41:13 PST

Primary and secondary structures of mammalian mitochondrial (mt) tRNAs are divergent from canonical tRNA structures due to highly skewed nucleotide content and large size variability of D- and T-loops. The nonconservation of nucleotides involved in the expected network of tertiary interactions calls into question the rules governing a functional L-shaped three-dimensional (3D) structure. Here, we report the solution structure of human mt-tRNA^Asp in its native post-transcriptionally modified form and as an in vitro transcript. Probing performed with nuclease S1, ribonuclease V1, dimethylsulfate, diethylpyrocarbonate and lead, revealed several secondary structures for the in vitro transcribed mt-tRNA^Asp including predominantly the cloverleaf. On the contrary, the native tRNA^Asp folds into a single cloverleaf structure, highlighting the contribution of the four newly identified post-transcriptional modifications to correct folding. Reactivities of nucleotides and phosphodiester bonds in the native tRNA favor existence of a full set of six classical tertiary interactions between the D-domain and the variable region, forming the core of the 3D structure. Reactivities of D- and T-loop nucleotides support an absence of interactions between these domains. According to multiple sequence alignments and search for conservation of Leontis–Westhof interactions, the tertiary network core building rules apply to all tRNA^Asp from mammalian mitochondria.

Translation of the FMR1 mRNA is not influenced by AGG interruptions

Fri, 13 Nov 2009 06:41:13 PST

The fragile X mental retardation 1 (FMR1) gene contains a CGG-repeat element within its 5' untranslated region (5'UTR) which, for alleles with more than ~40 repeats, increasingly affects both transcription (up-regulation) and translation (inhibition) of the repeat-containing RNA with increasing CGG-repeat length. Translational inhibition is thought to be due to impaired ribosomal scanning through the CGG-repeat region, which is postulated to form highly stable secondary/tertiary structure. One striking difference between alleles in the premutation range (55–200 CGG repeats) and those in the normal range (<~40 repeats) is the reduced number/absence of ‘expansion stabilizing’ AGG interruptions in the larger alleles. Such interruptions, which generally occur every 9–11 repeats in normal alleles, are thought to disrupt the extended CGG-repeat hairpin structure, thus facilitating translational initiation. To test this hypothesis, we have measured the translational efficiency of CGG-repeat mRNAs with 0–2 AGG interruptions, both in vitro (rabbit reticulocyte lysates) and in cell culture (HEK-293 cells). We demonstrate that the AGG interruptions have no detectable influence on translational efficiency in either a cell-free system or cell culture, indicating that any AGG-repeat-induced alterations in secondary/tertiary structure, if present, do not involve the rate-limiting step(s) in translational initiation.

Evidence for large diversity in the human transcriptome created by Alu RNA editing

Fri, 13 Nov 2009 06:41:13 PST

Adenosine-to-inosine (A-to-I) RNA editing alters the original genomic content of the human transcriptome and is essential for maintenance of normal life in mammals. A-to-I editing in Alu repeats is abundant in the human genome, with many thousands of expressed Alu sequences undergoing editing. Little is known so far about the contribution of Alu editing to transcriptome complexity. Transcripts derived from a single edited Alu sequence can be edited in multiple sites, and thus could theoretically generate a large number of different transcripts. Here we explored whether the combinatorial potential nature of edited Alu sequences is actually fulfilled in the human transcriptome. We analyzed datasets of editing sites and performed an analysis of a detailed transcript set of one edited Alu sequence. We found that editing appears at many more sites than detected by earlier genomic screens. To a large extent, editing of different sites within the same transcript is only weakly correlated. Thus, rather than finding a few versions of each transcript, a large number of edited variants arise, resulting in immense transcript diversity that eclipses alternative splicing as mechanism of transcriptome diversity, although with less impact on the proteome.

Recognition and coupling of A-to-I edited sites are determined by the tertiary structure of the RNA

Enstero, M., Daniel, C., Wahlstedt, H., Major, F., Ohman, M. — Fri, 13 Nov 2009 06:41:13 PST

Adenosine-to-inosine (A-to-I) editing has been shown to be an important mechanism that increases protein diversity in the brain of organisms from human to fly. The family of ADAR enzymes converts some adenosines of RNA duplexes to inosines through hydrolytic deamination. The adenosine recognition mechanism is still largely unknown. Here, to investigate it, we analyzed a set of selectively edited substrates with a cluster of edited sites. We used a large set of individual transcripts sequenced by the 454 sequencing technique. On average, we analyzed 570 single transcripts per edited region at four different developmental stages from embryogenesis to adulthood. To our knowledge, this is the first time, large-scale sequencing has been used to determine synchronous editing events. We demonstrate that edited sites are only coupled within specific distances from each other. Furthermore, our results show that the coupled sites of editing are positioned on the same side of a helix, indicating that the three-dimensional structure is key in ADAR enzyme substrate recognition. Finally, we propose that editing by the ADAR enzymes is initiated by their attraction to one principal site in the substrate.

Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 'Slicer' independent and can be mediated by Ago1

Fri, 13 Nov 2009 06:41:13 PST

It is known that siRNAs are capable of reducing expression of non-target genes due to the interaction of the siRNA guide strand with a partially complementary site on the ‘off-target’ mRNA. In the current study, we show that reduction of cellular Ago2 levels has no effect on off-target reduction of endogenous genes and that off-target degradation of mRNA can occur even in an Ago2 knockout cell line. Using antisense mediated reduction of Ago proteins and chemically modified cleavage- and binding-deficient siRNAs, we demonstrate that siRNA mediated off-target reduction is Ago2 cleavage independent, but does require siRNA interaction with either Ago1 or Ago2 and the RISC-loading complex. We also show that depletion of P-body associated proteins results in a reduction of off-target siRNA-mediated degradation of mRNA. Finally, we present data suggesting that a significant portion of on-target siRNA activity is also Ago2 cleavage independent, however, this activity does not appear to be P-body associated.

Recognition of tRNAGln by Helicobacter pylori GluRS2--a tRNAGln-specific glutamyl-tRNA synthetase

Chang, K.-M., Hendrickson, T. L. — Fri, 13 Nov 2009 06:41:14 PST

Accurate aminoacylation of tRNAs by the aminoacyl-tRNA synthetases (aaRSs) plays a critical role in protein translation. However, some of the aaRSs are missing in many microorganisms. Helicobacter pylori does not have a glutaminyl-tRNA synthetase (GlnRS) but has two divergent glutamyl-tRNA synthetases: GluRS1 and GluRS2. Like a canonical GluRS, GluRS1 aminoacylates tRNA^Glu1 and tRNA^Glu2. In contrast, GluRS2 only misacylates tRNA^Gln to form Glu-tRNA^Gln. It is not clear how GluRS2 achieves specific recognition of tRNA^Gln while rejecting the two H. pylori tRNA^Glu isoacceptors. Here, we show that GluRS2 recognizes major identity elements clustered in the tRNA^Gln acceptor stem. Mutations in the tRNA anticodon or at the discriminator base had little to no impact on enzyme specificity and activity.

The 5'-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex

Xu, N., Gkountela, S., Saeed, K., Akusjarvi, G. — Fri, 13 Nov 2009 06:41:14 PST

Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5'-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3'-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5'-strand of mivaRNAI. Of the small number of 5'-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3'-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5'-strand.

The fragment structure of a putative HsdR subunit of a type I restriction enzyme from Vibrio vulnificus YJ016: implications for DNA restriction and translocation activity

Uyen, N. T., Park, S.-Y., Choi, J.-W., Lee, H.-J., Nishi, K., Kim, J.-S. — Fri, 13 Nov 2009 06:41:14 PST

Among four types of bacterial restriction enzymes that cleave a foreign DNA depending on its methylation status, type I enzymes composed of three subunits are interesting because of their unique DNA cleavage and translocation mechanisms performed by the restriction subunit (HsdR). The elucidated N-terminal fragment structure of a putative HsdR subunit from Vibrio vulnificus YJ016 reveals three globular domains. The nucleolytic core within an N-terminal nuclease domain (NTD) is composed of one basic and three acidic residues, which include a metal-binding site. An ATP hydrolase (ATPase) site at the interface of two RecA-like domains (RDs) is located close to the probable DNA-binding site for translocation, which is far from the NTD nucleolytic core. Comparison of relative domain arrangements with other functionally related ATP and/or DNA complex structures suggests a possible translocation and restriction mechanism of the HsdR subunit. Furthermore, careful analysis of its sequence and structure implies that a linker helix connecting two RDs and an extended region within the nuclease domain may play a central role in switching the DNA translocation into the restriction activity.

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes

Fri, 13 Nov 2009 06:41:14 PST

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon–helix–helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes.

Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides

Gibson, D. G. — Fri, 13 Nov 2009 06:41:14 PST

Here it is demonstrated that the yeast Saccharomyces cerevisiae can take up and assemble at least 38 overlapping single-stranded oligonucleotides and a linear double-stranded vector in one transformation event. These oligonucleotides can overlap by as few as 20 bp, and can be as long as 200 nucleotides in length. This straightforward scheme for assembling chemically-synthesized oligonucleotides could be a useful tool for building synthetic DNA molecules.