Proteins In Higher Eukaryotes Research Articles

Substrate preference of protein kinase B isoforms can vary depending on the cell line.

Many proteins in higher eukaryotes, especially those with crucial functions, have multiple isoforms with redundant roles providing protection against potential functional deficiencies in one isoform. However, these isoforms can also have some unique roles. Protein kinase B, also known as Akt, is one such protein that has three isoforms encoded on different genes. Due to high sequence similarity and the general lack of specific reagents, most studies on Akt generalize their findings and do not distinguish between the isoforms. Using an established chemical genetic strategy and a set of known Akt substrates, this work explores substrate specificity of Akt isoforms under steady state conditions in two commonly used cell lines. This strategy can be applied to study any Akt isoform-specific substrates of interest in any cell line of choice as long as the cell line can be transfected.

Field model for multistate lateral diffusion of various transmembrane proteins observed in living Dictyostelium cells.

The lateral diffusion of transmembrane proteins on plasma membranes is a fundamental process for various cellular functions. Diffusion properties specific for individual protein species have been extensively studied, but the common features among protein species are poorly understood. Here, we systematically studied the lateral diffusion of various transmembrane proteins in the lower eukaryote Dictyostelium discoideum cells using a hidden Markov model for single-molecule trajectories obtained experimentally. As common features, all membrane proteins that had from one to ten transmembrane regions adopted three free diffusion states with similar diffusion coefficients regardless of their structural variability. All protein species reduced their mobility similarly upon the inhibition of microtubule or actin cytoskeleton dynamics, or myosin II. The relationship between protein size and the diffusion coefficient was consistent with the Saffman-Delbrück model, meaning that membrane viscosity is a major determinant of lateral diffusion, but protein size is not. These protein species-independent properties of multistate free diffusion were explained simply and quantitatively by free diffusion on the three membrane regions with different viscosities, which is in sharp contrast to the complex diffusion behavior of transmembrane proteins in higher eukaryotes.

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organization and function of RNA-protein complexes.

Large RNA-binding complexes play a central role in gene expression and orchestrate production, function, and turnover of mRNAs. The accuracy and dynamics of RNA–protein interactions within these molecular machines are essential for their function and are mediated by RNA-binding proteins (RBPs). Here, we show that fission yeast whole-cell poly(A)+ RNA–protein crosslinking data provide information on the organization of RNA–protein complexes. To evaluate the relative enrichment of cellular RBPs on poly(A)+ RNA, we combine poly(A)+ RNA interactome capture with a whole-cell extract normalization procedure. This approach yields estimates of in vivo RNA-binding activities that identify subunits within multiprotein complexes that directly contact RNA. As validation, we trace RNA interactions of different functional modules of the 3′ end processing machinery and reveal additional contacts. Extending our analysis to different mutants of the RNA exosome complex, we explore how substrate channeling through the complex is affected by mutation. Our data highlight the central role of the RNA helicase Mtl1 in regulation of the complex and provide insights into how different components contribute to engagement of the complex with substrate RNA. In addition, we characterize RNA-binding activities of novel RBPs that have been recurrently detected in the RNA interactomes of multiple species. We find that many of these, including cyclophilins and thioredoxins, are substoichiometric RNA interactors in vivo. Because RBPomes show very good overall agreement between species, we propose that the RNA-binding characteristics we observe in fission yeast are likely to apply to related proteins in higher eukaryotes as well.

Selectivity in agonist and antagonist binding to Serotonin1A receptors via G-protein coupling

G protein-coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins in higher eukaryotes, and facilitate information transfer from the extracellular environment to the cellular interior upon activation by ligands. Their role in diverse signaling processes makes them an attractive choice as drug targets. GPCRs are coupled to heterotrimeric G-proteins which represent an important interface through which signal transduction occurs across the plasma membrane upon activation by ligands. To obtain further insight into the molecular details of interaction of G-proteins with GPCRs, in this work, we explored the selectivity of binding of specific agonists and antagonists to the serotonin1A receptor under conditions of progressive G-protein inactivation. The serotonin1A receptor is an important neurotransmitter receptor belonging to the GPCR family and is a popular drug target. By use of a number of agents to inactivate G-proteins, we show here that the serotonin1A receptor displays differential discrimination between agonist and antagonist binding. Our results show a reduction in binding sites of the receptor upon treatment with G-protein inactivating agents. In addition, G-protein coupling efficiency was enhanced when G-proteins were inactivated using urea and alkaline pH. We envision that our results could be useful in achieving multiple signaling states of the receptor by fine tuning the conditions of G-protein inactivation and in structural biology of GPCRs bound to specific ligands.

Role of Actin Cytoskeleton in Dynamics and Function of the Serotonin1A Receptor

G protein-coupled receptors (GPCRs) are important membrane proteins in higher eukaryotes that carry out a vast array of cellular signaling and act as major drug targets. The serotonin1A receptor is a prototypical member of the GPCR family and is implicated in neuropsychiatric disorders such as anxiety and depression, besides serving as an important drug target. With an overall goal of exploring the functional consequence of altered receptor dynamics, in this work, we probed the role of the actin cytoskeleton in the dynamics, ligand binding, and signaling of the serotonin1A receptor. We monitored receptor dynamics utilizing single particle tracking, which provides information on relative distribution of receptors in various diffusion modes in addition to diffusion coefficient. We show here that the short-term diffusion coefficient of the receptor increases upon actin destabilization by cytochalasin D. In addition, analysis of individual trajectories shows that there are changes in relative populations of receptors undergoing various types of diffusion upon actin destabilization. The release of dynamic constraint was evident by an increase in the radius of confinement of the receptor upon actin destabilization. The functional implication of such actin destabilization was manifested as an increase in specific agonist binding and downstream signaling, monitored by measuring reduction in cellular cAMP levels. These results bring out the interdependence of GPCR dynamics with cellular signaling.

Lin28 promotes dental pulp cell proliferation via upregulation of cyclin-dependent proteins and interaction with let-7a/IGF2BP2 pathways

Caries, pulpitis, and trauma are the main causes of dental pulp damage. The regeneration capacity of dental pulp declines with age. Lin28 is a conserved RNA-binding protein in higher eukaryotes that regulates several important cellular functions associated with development, glucose metabolism, differentiation, and pluripotency. Conditional reactivation of Lin28 gene in adult mice markedly accelerates the wound-healing process in injured digits. However, little is known about its functions and molecular mechanism in human dental pulp. The aim of this study was to investigate the effects and mechanism of overexpression of Lin28 gene on the proliferation of human dental pulp cells (HDPCs). For this purpose, a number of molecular and biochemical analytical techniques, including the ethynyl-2ʹ-deoxyuridine (EdU) incorporation assay, RNA-protein immunoprecipitation (RIP) analysis, and luciferase assays, were used for detailed characterization. In addition, factors regulating HDPCs activation were explored through gain-of-function and loss-of-function analyses. The results demonstrate that Lin28 promotes cell proliferation and the S-G2/M transition of HDPCs and directly binds to a group of cell cycle regulatory mRNAs in HDPCs. Through bioinformatics analysis and luciferase assays, we confirmed that let-7a targets IGF2BP2. Silencing of IGF2BP2 showed similar cellular and molecular effects as let-7a. Similarly, restoration of IGF2BP2 counteracted the effects of let-7a expression. In conclusion, Lin28 promotes cell proliferation by regulation of both mRNA translation (let-7-independent) and miRNA biogenesis (let-7-dependent). Lin28 can promote the expression of pro-proliferative genes by directly enhancing their translation to maintain a tight control over HDPC proliferation.

The heterotrimeric G protein г Stgg1 is required for conidiation, secondary metabolite production and pathogenicity of Setosphaeria turcica

ABSTRACTHeterotrimeric G proteins are best known for their role in the transduction of extracellular signals to various downstream effectors. G proteins in higher eukaryotes are intensively studied; however, their roles in foliar pathogens are still elusive. In this study, we cloned the gene Stgg1 encoding G protein γ subunit in Setosphaeria turcica and investigated its function by RNA interference technology. Three independent Stgg1 targeted RNAi mutants R3, R5 and R6 with diverse silencing efficiency were generated. Knock-down of Stgg1 resulted in a significant reduction in mRNA levels of the genes encoding Gα (Stga1, Stga2, Stga3) but not for Gβ (Stgb1). Stgg1 RNAi mutants exhibited significantly elongated hyphal cells with blocked conidium production. In addition, Stgg1 RNAi mutants all appeared in lighter colony colour compatible with inhibited secondary metabolites. Further assays demonstrated that Stgg1 was required for biosynthesis of melanin and HT-toxin activity. Furthermore, down-regulation of Stgg1 largely inhibited the inflection capacity. Thus, we proposed that Stgg1 played crucial roles in conidiation, secondary metabolite production and pathogenicity of S. turcica and is, therefore, an ideal target for drug design against foliar pathogens.

Biochemical and Biophysical Characterisation of Higher Oligomeric Structure of Rat Nucleosome Assembly Protein 1.

Nucleosome assembly protein 1 (NAP1) is a histone chaperone that exchanges histone H2A-H2B dimer from chromatin templates. Studies with yeast NAP1 (yNAP1) have revealed itsexistence asmultiple oligomeric species in solution. Here, rat NAP1 (rNAP1), which is 98% identical to the human NAP1 (hNAP1) was used as a model to characterize the oligomeric structures of this protein in higher eukaryotes. Gel filtration chromatography and Dynamic light scattering of recombinant rNAP1 indicated that the protein exists as a complex mixture of multimeric species even at 500mM ionic strength. The solution-state complexity remains unchanged even at higher ionic strengths. Equilibrium unfolding (ΔG 14.6kcalmol- 1) shows that rNAP1, both dimeric and oligomeric, follow the two-state model of unfolding with no detectable intermediates. Homology modelling suggests that rat and yeast NAP1 share an overall similar structure with conserved domains. However, dissimilar substitutions like threonine and lysine with glycine in the β-hairpin involved in oligomerization, possibly leads to the observed differences in the oligomerization propensity of the two proteins. Molecular dynamic simulation (MDS) of the two structures also revealed that rNAP1 dimer is more stable owing to the extensive hydrogen bonding in comparison to yNAP1. Further, in vitro kinase assay showed that the phosphorylation of rNAP1 favors oligomerization with no effect on its histone binding capacity. Our results clearly suggest that there are differences in the in-solution behavior of rNAP1 compared to yNAP1 which may have in vivo functional implications for the regulation of these complexes during chromatin assembly and rearrangement.

Temperature and osmotic stress dependence of the thermodynamics for binding linker histone H10, Its carboxyl domain (H10-C) or globular domain (H10-G) to B-DNA

Linker histones (H1) are the basic proteins in higher eukaryotes that are responsible for the final condensation of chromatin. In contrast to the nucleosome core histone proteins, the role of H1 in compacting DNA is not clearly understood. In this study ITC was used to measure the binding constant, enthalpy change, and binding site size for the interactions of H10, or its C-terminal (H10-C) and globular (H10-G) domains to highly polymerized calf-thymus DNA at temperatures from 288K to 308K. Heat capacity changes, ΔCp, for these same H10 binding interactions were estimated from the temperature dependence of the enthalpy changes. The enthalpy changes for binding H10, H10-C, or H10-G to CT-DNA are all endothermic at 298K, becoming more exothermic as the temperature is increased. The ΔH for binding H10-G to CT-DNA is exothermic at temperatures above approximately 300K. Osmotic stress experiments indicate that the binding of H10 is accompanied by the release of approximately 35 water molecules.We estimate from our naked DNA titration results that the binding of the H10 to the nucleosome places the H10 protein in close contact with approximately 41 DNA bp. The breakdown is that the H10 carboxyl terminus interacts with 28bp of linker DNA on one side of the nucleosome, the H10 globular domain binds directly to 7bp of core DNA, and shields another 6 linker DNA bases, 3bp on either side of the nucleosome where the linker DNA exits the nucleosome core.

The Role of the Trypanosoma cruzi TcNRBD1 Protein in Translation.

The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. Despite the importance of this type of control in Trypanosoma cruzi, few RNA binding proteins have been characterized. The RRM domain (RNA Recognition Motif) is one of the most abundant domains found in RNA-binding proteins in higher eukaryotes. Proteins containing the RRM domain are involved in the majority of post-transcriptional processes regulating gene expression. In this work, we aimed to characterize the protein TcNRBD1 from T. cruzi. TcNRBD1 is an RNA-binding protein that contains 2 RRM domains and is the ortholog of the P34 and P37 proteins from Trypanosoma brucei. The TcNRBD1 protein is expressed in all developmental stages of T. cruzi, and its localization pattern is concentrated at the perinuclear region. TcNRBD1 is associated with polysomes and with the 80S monosomes. Furthermore, sequencing of the mRNAs bound to TcNRBD1 allowed the identification of several transcripts that encode ribosomal proteins. Immunoprecipitation assays followed by mass spectrometry showed that the protein complexes with several ribosomal proteins from both the 40S and 60S subunits. In summary, the results indicate that TcNRBD1 is associated with different parts of the translation process, either by regulating mRNAs that encode ribosomal proteins or by acting in some step of ribosome assembly in T. cruzi.

The Exonuclease Homolog OsRAD1 Promotes Accurate Meiotic Double-Strand Break Repair by Suppressing Nonhomologous End Joining.

During meiosis, programmed double-strand breaks (DSBs) are generated to initiate homologous recombination, which is crucial for faithful chromosome segregation. In yeast, Radiation sensitive1 (RAD1) acts together with Radiation sensitive9 (RAD9) and Hydroxyurea sensitive1 (HUS1) to facilitate meiotic recombination via cell-cycle checkpoint control. However, little is known about the meiotic functions of these proteins in higher eukaryotes. Here, we characterized a RAD1 homolog in rice (Oryza sativa) and obtained evidence that O. sativa RAD1 (OsRAD1) is important for meiotic DSB repair. Loss of OsRAD1 led to abnormal chromosome association and fragmentation upon completion of homologous pairing and synapsis. These aberrant chromosome associations were independent of OsDMC1. We found that classical nonhomologous end-joining mediated by Ku70 accounted for most of the ectopic associations in Osrad1 In addition, OsRAD1 interacts directly with OsHUS1 and OsRAD9, suggesting that these proteins act as a complex to promote DSB repair during rice meiosis. Together, these findings suggest that the 9-1-1 complex facilitates accurate meiotic recombination by suppressing nonhomologous end-joining during meiosis in rice.

Arabidopsis AtMORC4 and AtMORC7 Form Nuclear Bodies and Repress a Large Number of Protein-Coding Genes.

The MORC family of GHKL ATPases are an enigmatic class of proteins with diverse chromatin related functions. In Arabidopsis, AtMORC1, AtMORC2, and AtMORC6 act together in heterodimeric complexes to mediate transcriptional silencing of methylated DNA elements. Here, we studied Arabidopsis AtMORC4 and AtMORC7. We found that, in contrast to AtMORC1,2,6, they act to suppress a wide set of non-methylated protein-coding genes that are enriched for those involved in pathogen response. Furthermore, atmorc4 atmorc7 double mutants show a pathogen response phenotype. We found that AtMORC4 and AtMORC7 form homomeric complexes in vivo and are concentrated in discrete nuclear bodies adjacent to chromocenters. Analysis of an atmorc1,2,4,5,6,7 hextuple mutant demonstrates that transcriptional de-repression is largely uncoupled from changes in DNA methylation in plants devoid of MORC function. However, we also uncover a requirement for MORC in both DNA methylation and silencing at a small but distinct subset of RNA-directed DNA methylation target loci. These regions are characterized by poised transcriptional potential and a low density of sites for symmetric cytosine methylation. These results provide insight into the biological function of MORC proteins in higher eukaryotes.

Solubilization and Characterization of β-Mannosyltransferase from Pig Aorta

Glycosyltransferase in general play a very important role in the post-translational modification of number of biomolecules including glycoproteins. The glycosylation of the protein occurs in the endoplasmic reticulum and is carried out by at least 14 different types of membrane bound glycosyltransferases. It is now well established that glycosylation of protein occurs either at serine or threonine residues (O-glycosylation) or at asparagine residue (N-glycosylation) of proteins. To date there is no definite demonstration of the presence of O-mannosylation of proteins in higher eukaryotes. However, it is a common feature in yeast and other fungal strain, indicating that glycosyltransferases are species-specific as well as tissue-specific. In higher eukaryotes the Nglycosylation is the major post translational process, initiated by the synthesis of a large lipid oligosaccharide, Glc3-Man9-GlcNAc2-PP-Dolichol. This represents the immediate precursor of carbohydrate unit involved in the biosynthesis of glycoproteins. The large oligosaccharide is then transferred from the lipid oligosaccharide to the acceptor lipid by the oligosaccharide transferase. β-Mannosyltransferase is the key enzyme of the dolichol pathway that catalyzes the transfer of mannose to the chitobiose lipid (GlcNAc2-PP-Dol), acceptor substrate from GDP-mannose. This enzyme has been purified to near homogeneity and characterized from pig aorta tissue, using different chromatography techniques, and studying its properties.

Distinct Roles of Alpha/Beta Hydrolase Domain Containing Proteins

Lipid molecules are not only serve as building blocks of biological membranes but are also progressively documented for their role in various biological processes. Alpha/beta hydrolase domain (ABHD) containing proteins are structurally related with diverse catalytic activities. Members in this family include various enzymes with broad substrate specificities. Proteins from these families are having a conserved ABHD region and lipase and acyltransferase motifs. However, little is known about ABHD proteins in plants. This article discusses about the biological roles and the implications of ABHD domain containing proteins in higher eukaryotes.

A possible role of Drosophila CTCF in mitotic bookmarking and maintaining chromatin domains during the cell cycle.

BackgroundThe CCCTC-binding factor (CTCF) is a highly conserved insulator protein that plays various roles in many cellular processes. CTCF is one of the main architecture proteins in higher eukaryotes, and in combination with other architecture proteins and regulators, also shapes the three-dimensional organization of a genome. Experiments show CTCF partially remains associated with chromatin during mitosis. However, the role of CTCF in the maintenance and propagation of genome architectures throughout the cell cycle remains elusive.ResultsWe performed a comprehensive bioinformatics analysis on public datasets of Drosophila CTCF (dCTCF). We characterized dCTCF-binding sites according to their occupancy status during the cell cycle, and identified three classes: interphase-mitosis-common (IM), interphase-only (IO) and mitosis-only (MO) sites. Integrated function analysis showed dCTCF-binding sites of different classes might be involved in different biological processes, and IM sites were more conserved and more intensely bound. dCTCF-binding sites of the same class preferentially localized closer to each other, and were highly enriched at chromatin syntenic and topologically associating domains boundaries.ConclusionsOur results revealed different functions of dCTCF during the cell cycle and suggested that dCTCF might contribute to the establishment of the three-dimensional architecture of the Drosophila genome by maintaining local chromatin compartments throughout the whole cell cycle.Electronic supplementary materialThe online version of this article (doi:10.1186/s40659-015-0019-6) contains supplementary material, which is available to authorized users.

Expression, purification and crystallization of the FP domain of the human F-box protein Fbxo7

Fbxo7 is a conserved protein in higher eukaryotes that belongs to the F-box protein family. Fbxo7 is the substrate-recognition component of the SCFFbxo7 (Skp1-Cul1-Fbox protein) E3 ubiquitin ligase. Besides the F-box motif, Fbxo7 also contains a C-terminal proline-rich region, an N-terminal ubiquitin-like domain and a novel FP (Fbxo7/PI31) domain preceding the F-box motif. The FP domains of Fbxo7 and the PI31 proteasome inhibitor mediate interaction between the two proteins. For structure determination of the FP domain of Fxbo7, a protein construct (amino acids 181-335) corresponding to the FP domain was expressed, purified and crystallized. The native and selenomethionine-labeled proteins crystallized in different crystal forms. Native and single-wavelength anomalous dispersion data sets with diffraction to 2.1 and 2.0 Å resolution, respectively, have been collected and structure determination is in progress.

Global proteomic analysis in trypanosomes reveals unique proteins and conserved cellular processes impacted by arginine methylation

Arginine methylation is a common posttranslational modification with reported functions in transcription, RNA processing and translation, and DNA repair. Trypanosomes encode five protein arginine methyltransferases, suggesting that arginine methylation exerts widespread impacts on the biology of these organisms. Here, we performed a global proteomic analysis of Trypanosoma brucei to identify arginine methylated proteins and their sites of modification. Using an approach entailing two-dimensional chromatographic separation and alternating electron transfer dissociation and collision induced dissociation, we identified 1332 methylarginines in 676 proteins. The resulting data set represents the largest compilation of arginine methylated proteins in any organism to date. Functional classification revealed numerous arginine methylated proteins involved in flagellar function, RNA metabolism, DNA replication and repair, and intracellular protein trafficking. Thus, arginine methylation has the potential to impact aspects of T. brucei gene expression, cell biology, and pathogenesis. Interestingly, pathways with known methylated proteins in higher eukaryotes were identified in this study, but often different components of the pathway were methylated in trypanosomes. Methylarginines were often identified in glycine rich contexts, although exceptions to this rule were detected. Collectively, these data inform on a multitude of aspects of trypanosome biology and serve as a guide for the identification of homologous arginine methylated proteins in higher eukaryotes. T. brucei is a protozoan parasite that causes lethal African sleeping sickness in humans and nagana in livestock, thereby imposing a significant medical and economic burden on sub-Saharan Africa. The parasite encounters very different environments as it cycles between mammalian and insect hosts, and must exert cellular responses to these varying milieus. One mechanism by which all cells respond to changing environments is through posttranslational modification of proteins. Arginine methylation is one such modification that can dramatically impact protein-protein and protein-nucleic acid interactions and subcellular localization of proteins. To define the breadth of arginine methylation in trypanosomes and identify target proteins, we performed a global proteomic analysis of arginine methylated proteins in insect stage T. brucei. We identified 1332 methylarginines in 676 proteins, generating the largest compilation of methylarginine containing proteins in any organism to date. Numerous arginine methylated proteins function in RNA and DNA related processes, suggesting this modification can impact T. brucei genome integrity and gene regulation at numerous points. Other processes that appear to be strongly influenced by arginine methylation are intracellular protein trafficking, signaling, protein folding and degradation, and flagellar function. The widespread nature of arginine methylation in trypanosomes highlights its potential to greatly affect parasite biology and pathogenesis.

One Single Basic Amino Acid at the ω-1 or ω-2 Site Is a Signal That Retains Glycosylphosphatidylinositol-Anchored Protein in the Plasma Membrane of Aspergillus fumigatus

Although the plasma membrane is the terminal destination for glycosylphosphatidylinositol (GPI) proteins in higher eukaryotes, cell wall-attached GPI proteins (GPI-CWPs) are found in many fungal species. In yeast, some of the cis-requirements directing localization of GPI proteins to the plasma membrane or cell wall are now understood. However, it remains to be determined how Aspergillus fumigatus, an opportunistic fungal pathogen, signals, and sorts GPI proteins to either the plasma membrane or the cell wall. In this study, chimeric green fluorescent proteins (GFPs) were constructed as fusions with putative C-terminal GPI signal sequences from A. fumigatus Mp1p, Gel1p, and Ecm33p, as well as site-directed mutations thereof. By analyzing cellular localization of chimeric GFPs using Western blotting, electron microscopy, and fluorescence microscopy, we showed that, in contrast to yeast, a single Lys residue at the ω-1 or ω-2 site alone could retain GPI-anchored GFP in the plasma membrane. Although the signal for cell wall distribution has not been identified yet, it appeared that the threonine/serine-rich region at the C-terminal half of AfMp1 was not required for cell wall distribution. Based on our results, the cis-requirements directing localization of GPI proteins in A. fumigatus are different from those in yeast.

Histone H1 Plays a Role in Heterochromatin Formation and VSG Expression Site Silencing in Trypanosoma brucei

The African sleeping sickness parasite Trypanosoma brucei evades the host immune system through antigenic variation of its variant surface glycoprotein (VSG) coat. Although the T. brucei genome contains ∼1500 VSGs, only one VSG is expressed at a time from one of about 15 subtelomeric VSG expression sites (ESs). For antigenic variation to work, not only must the vast VSG repertoire be kept silent in a genome that is mainly constitutively transcribed, but the frequency of VSG switching must be strictly controlled. Recently it has become clear that chromatin plays a key role in silencing inactive ESs, thereby ensuring monoallelic expression of VSG. We investigated the role of the linker histone H1 in chromatin organization and ES regulation in T. brucei. T. brucei histone H1 proteins have a different domain structure to H1 proteins in higher eukaryotes. However, we show that they play a key role in the maintenance of higher order chromatin structure in bloodstream form T. brucei as visualised by electron microscopy. In addition, depletion of histone H1 results in chromatin becoming generally more accessible to endonucleases in bloodstream but not in insect form T. brucei. The effect on chromatin following H1 knock-down in bloodstream form T. brucei is particularly evident at transcriptionally silent ES promoters, leading to 6–8 fold derepression of these promoters. T. brucei histone H1 therefore appears to be important for the maintenance of repressed chromatin in bloodstream form T. brucei. In particular H1 plays a role in downregulating silent ESs, arguing that H1-mediated chromatin functions in antigenic variation in T. brucei.

Atl1 Regulates Choice between Global Genome and Transcription-Coupled Repair of O6-Alkylguanines

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER.