Niels Erik Møllegaard

Polyamines preferentially interact with bent adenine tracts in double-stranded DNA

Polyamines, such as putrescine, spermidine and spermine, have indirectly been linked with the regulation of gene expression, and their concentrations are typically increased in cancer cells. Although effects on transcription factor binding to cognate DNA targets have been demonstrated, the mechanisms of the biological action of polyamines is poorly understood. Employing uranyl photo-probing we now demonstrate that polyamines at submillimolar concentrations bind preferentially to bent adenine tracts in double-stranded DNA. These results provide the first clear evidence for the sequence-specific binding of polyamines to DNA, and thereby suggest a mechanism by which the cellular effects of polyamines in terms of differential gene transcriptional activity could, at least partly, be a direct consequence of sequence-specific interactions of polyamines with promoters at the DNA sequence level.

DNA binding by the plant-specific NAC transcription factors in crystal and solution: a firm link to WRKY and GCM transcription factors.

NAC (NAM/ATAF/CUC) plant transcription factors regulate essential processes in development, stress responses and nutrient distribution in important crop and model plants (rice, Populus, Arabidopsis), which makes them highly relevant in the context of crop optimization and bioenergy production. The structure of the DNA-binding NAC domain of ANAC019 has previously been determined by X-ray crystallography, revealing a dimeric and predominantly β-fold structure, but the mode of binding to cognate DNA has remained elusive. In the present study, information from low resolution X-ray structures and small angle X-ray scattering on complexes with oligonucleotides, mutagenesis and (DNase I and uranyl photo-) footprinting, is combined to form a structural view of DNA-binding, and for the first time provide experimental evidence for the speculated relationship between plant-specific NAC proteins, WRKY transcription factors and the mammalian GCM (Glial cell missing) transcription factors, which all use a β-strand motif for DNA-binding. The structure shows that the NAC domain inserts the edge of its core β-sheet into the major groove, while leaving the DNA largely undistorted. The structure of the NAC-DNA complex and a new crystal form of the unbound NAC also indicate limited flexibility of the NAC dimer arrangement, which could be important in recognizing suboptimal binding sites.

Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence

Highlight HvNAC005 was shown to be a strong positive regulator of senescence, involved in regulation in the cross field of different hormone and signalling pathways controlling developmental senescence in barley.

A DNA minor groove electronegative potential genome map based on photo-chemical probing.

The double-stranded DNA of the genome contains both sequence information directly relating to the protein and RNA coding as well as functional and structural information relating to protein recognition. Only recently is the importance of DNA shape in this recognition process being fully appreciated, and it also appears that minor groove electronegative potential may contribute significantly in guiding proteins to their cognate binding sites in the genome. Based on the photo-chemical probing results, we have derived an algorithm that predicts the minor groove electronegative potential in a DNA helix of any given sequence. We have validated this model on a series of protein–DNA binding sites known to involve minor groove electrostatic recognition as well as on stable nucleosome core complexes. The algorithm allows for the first time a full minor groove electrostatic description at the nucleotide resolution of any genome, and it is illustrated how such detailed studies of this sequence dependent, inherent property of the DNA may reflect on genome organization, gene expression and chromosomal condensation.

GAGE Cancer-Germline Antigens Are Recruited to the Nuclear Envelope by Germ Cell-Less (GCL)

GAGE proteins are highly similar, primate-specific molecules with unique primary structure and undefined cellular roles. They are restricted to cells of the germ line in adult healthy individuals, but are broadly expressed in a wide range of cancers. In a yeast two-hybrid screen we identified the metazoan transcriptional regulator, Germ cell-less (GCL), as an interaction partner of GAGE12I. GCL directly binds LEM-domain proteins (LAP2β, emerin, MAN1) at the nuclear envelope, and we found that GAGE proteins were recruited to the nuclear envelope inner membrane by GCL. Based on yeast two-hybrid analysis and pull-down experiments of GCL polypeptides, GCL residues 209–320 (which includes the BACK domain) were deduced sufficient for association with GAGE proteins. GAGE mRNAs and GCL mRNA were demonstrated in human testis and most types of cancers, and at the protein level GAGE members and GCL were co-expressed in cancer cell lines. Structural studies of GAGE proteins revealed no distinct secondary or tertiary structure, suggesting they are intrinsically disordered. Interestingly GAGE proteins formed stable complexes with dsDNA in vitro at physiological concentrations, and GAGE12I bound several different dsDNA fragments, suggesting sequence-nonspecific binding. Dual association of GAGE family members with GCL at the nuclear envelope inner membrane in cells, and with dsDNA in vitro, implicate GAGE proteins in chromatin regulation in germ cells and cancer cells.

Dissecting direct and indirect readout of cAMP receptor protein DNA binding using an inosine and 2,6-diaminopurine in vitro selection system

The DNA interaction of the Escherichia coli cyclic AMP receptor protein (CRP) represents a typical example of a dual recognition mechanism exhibiting both direct and indirect readout. We have dissected the direct and indirect components of DNA recognition by CRP employing in vitro selection of a random library of DNA-binding sites containing inosine (I) and 2,6-diaminopurine (D) instead of guanine and adenine, respectively. Accordingly, the DNA helix minor groove is structurally altered due to the ‘transfer’ of the 2-amino group of guanine (now I) to adenine (now D), whereas the major groove is functionally intact. The majority of the selected sites contain the natural consensus sequence TGTGAN6TCACA (i.e. TITIDN6TCDCD). Thus, direct readout of the consensus sequence is independent of minor groove conformation. Consequently, the indirect readout known to occur in the TG/CA base pair step (primary kink site) in the consensus sequence is not affected by I–D substitutions. In contrast, the flanking regions are selected as I/C rich sequences (mostly I-tracts) instead of A/T rich sequences which are known to strongly increase CRP binding, thereby demonstrating almost exclusive indirect readout of helix structure/flexibility in this region through (anisotropic) flexibility of I-tracts.

SSX2 is a novel DNA-binding protein that antagonizes polycomb group body formation and gene repression

Polycomb group (PcG) complexes regulate cellular identity through epigenetic programming of chromatin. Here, we show that SSX2, a germline-specific protein ectopically expressed in melanoma and other types of human cancers, is a chromatin-associated protein that antagonizes BMI1 and EZH2 PcG body formation and derepresses PcG target genes. SSX2 further negatively regulates the level of the PcG-associated histone mark H3K27me3 in melanoma cells, and there is a clear inverse correlation between SSX2/3 expression and H3K27me3 in spermatogenesis. However, SSX2 does not affect the overall composition and stability of PcG complexes, and there is no direct concordance between SSX2 and BMI1/H3K27me3 presence at regulated genes. This suggests that SSX2 antagonizes PcG function through an indirect mechanism, such as modulation of chromatin structure. SSX2 binds double-stranded DNA in a sequence non-specific manner in agreement with the observed widespread association with chromatin. Our results implicate SSX2 in regulation of chromatin structure and function.

Phosphate Selective Uranyl Photo‐Affinity Cleavage of Proteins. Determination of Phosphorylation Sites

Phosphorylation of proteins is one of the most important mechanisms in cellular signaling and is involved in cellular processes such as metabolism, transcription, translation, cell cycle, movement, apoptosis, and differentiation. Phosphorylation takes place at serine, threonine, and tyrosine residues in a 1000:100:1 ratio, and it has been estimated that 30% of all proteins are reversibly phosphorylated at one or multiple sites at some point during their lifetime. This is facilitated by the high number of kinases and phosphatases, which constitute 2% of all human genome genes. Phosphorylation of proteins is a reversible process, and proteins can be phosphorylated at substoichiometric levels. For proteins containing multiple phosphorylation sites, each site can be associated with a different function; this makes the particular function in question ACHTUNGTRENNUNGdependent on the phosphorylation pattern. To understand the role of dynamic protein phosphorylation, the identification of the exact sites and extent of phosphorylation is crucial. At present, state-of-the-art techniques for investigation of protein phosphorylation make use of mass spectrometry (MS), subsequent to protease digests (most often trypsin) of an isolated phosphoprotein or of the entire phosphoproteome in a cell lysate. Secondly, phosphopeptide enrichment is carried out to reduce the number of peptides to be analyzed. Finally N-terminal sequencing and/or MS analysis are performed to identify the specific positions of phosphorylation. Although different MS approaches have been used for detecting phosphorylated positions in the proteome, limitations and difficulties remain concerning signal suppression of phosphate containing peptides, dephosphorylation, difficulties in achieving coverage of the full length of long peptides, peptides present in low amounts, peptides phosphorylated at substoichiometric levels, and finally, difficulties in distinguishing among multiple possible phosphorylation sites within in a given peptide fragment. Thus, analysis of peptides after trypsin digests is not straightforward and determination of the exact site of phosphorylation often fails for recovered phosphopepACHTUNGTRENNUNGtides. Therefore alternative approaches have been considered in order to develop improved methods for phosphorylation site determination in the proteome. An attractive goal has been to develop phosphospecific proteases in order to reduce the amount of peptide products, which have to be analyzed. Furthermore, such a protease would generate peptide products containing the phosphoamino acid residue positioned either at the N or C terminus thereby significantly simplifying MS analysis and the identification of the phosphorylation sites. Although no phosphospecific protease has so far been found in nature, a chemical approach has been developed where phosphoserines and phosphothreonines are converted into lysine analogues (amino-ethyl cysteine and b-methyl S-ethyl cysteine) to generate cleavage sites for lysine specific proteases. However, this methodology is both complex and laborious and involves several chemical and enzymatic modifications, and most importantly, induces problems in distinguishing naturally occurring lysines from those generated from phosphorylated serine and threonine residues. Uranyl photocleavage has for two decades been used for studying protein–double-stranded (ds) DNA interactions, drug– dsDNA interactions, and the interactions of metal-ions with nucleic acids. Uranyl photocleavage of nucleic acids is based on the high affinity of the divalent uranyl cation for phosphates in the backbone of nucleic acids and the strong oxidation power of the excited state of the uranyl ion. Upon excitation of the bound uranyl ion, the high oxidation potential of uranyl induces breakage at the sugar-phosphate backbone at each nucleotide. It was recently reported that proteins can also be photocleaved by uranyl but at low efficiency. In view of the high affinity of the uranyl(VI) ion for phosphate, we have now systematically analyzed whether phosphorylated sites present in proteins could recruit the uranyl ion for subsequent cleavage. We find that both the specificity as well as the efficiency of the photocleavage reaction is increased in the presence of phosphorylated residues and this opens the potential application for detection of phosphorylation sites in proteins by uranyl photocleavage. To examine whether site-specific cleavage at phoshorylated residues in proteins could be induced by uranyl photocleavage, three different phosphoproteins were selected as model systems: a-casein, b-casein, and ovalbumin. The three proteins differ in size, structure, and extent of phosphorylation, and represent different patterns of phosphorylation in proteins. Initially all three proteins were subjected to uranyl photocleavage at different uranyl/protein ratios and irradiated at 320 nm. The [a] L. H. Kristensen, Prof. P. E. Nielsen, N. E. Møllegaard Department of Cellular and Molecular Medicine Panum Institute, University of Copenhagen Blegdamsvej 3, 2200 Copenhagen N (Denmark) Fax: (+45)35-32-77-32 E-mail : nem@imbg.ku.dk [b] C. I. Jørgensen Novozymes A/S Novo Alle 6B 3.90, 2880 Bagsværd (Denmark) [c] B. B. Kragelund Department of Biology, Structural Biology and NMR Laboratory University of Copenhagen Ole Maaløes Vej 5, 2200 Copenhagen N (Denmark) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

A phosphorylation tag for uranyl mediated protein purification and photo assisted tag removal.

Most protein purification procedures include an affinity tag fused to either the N or C-terminal end of the protein of interest as well as a procedure for tag removal. Tag removal is not straightforward and especially tag removal from the C-terminal end is a challenge due to the characteristics of enzymes available for this purpose. In the present study, we demonstrate the utility of the divalent uranyl ion in a new procedure for protein purification and tag removal. By employment of a GFP (green florescence protein) recombinant protein we show that uranyl binding to a phosphorylated C-terminal tag enables target protein purification from an E. coli extract by immobilized uranyl affinity chromatography. Subsequently, the tag can be efficiently removed by UV-irradiation assisted uranyl photocleavage. We therefore suggest that the divalent uranyl ion (UO2 2+) may provide a dual function in protein purification and subsequent C-terminal tag removal procedures.

A novel indirect sequence readout component in the E. coli cyclic AMP receptor protein operator.

The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNNNNTCACANTT through direct amino acid nucleobase interactions in the major groove of the two operator half-sites. Crystal structure analyses have revealed that the interaction results in two strong kinks at the TG/CA steps closest to the 6-base-pair spacer (N6). This spacer exhibits high sequence variability among the more than 100 natural binding sites in the E. coli genome, but the exact role of the N6 region in CRP interaction has not previously been systematic examined. Here we employ an in vitro selection system based on a randomized N6 spacer region to demonstrate that CRP binding to the lacP1 site may be enhanced up to 14-fold or abolished by varying the N6 spacer sequences. Furthermore, on the basis of sequence analysis and uranyl (UO2(2+)) probing data, we propose that the underlying mechanism relies on N6 deformability.