Journal of Molecular Biology

ScienceDirect RSS
  • Editorial Board
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17









    Categories: Journal Articles
  • Contents List
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17









    Categories: Journal Articles
  • Structure of Kif14: An Engaging Molecular Motor
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Sarah Rice







    Categories: Journal Articles
  • KIF14 Binds Tightly to Microtubules and Adopts a Rigor-Like Conformation
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Kritica Arora , Lama Talje , Ana B. Asenjo , Parker Andersen , Kaleem Atchia , Monika Joshi , Hernando Sosa , John S. Allingham , Benjamin H. Kwok

    The mitotic kinesin motor protein KIF14 is essential for cytokinesis during cell division and has been implicated in cerebral development and a variety of human cancers. Here we show that the mouse KIF14 motor domain binds tightly to microtubules and does not display typical nucleotide-dependent changes in this affinity. It also has robust ATPase activity but very slow motility. A crystal structure of the ADP-bound form of the KIF14 motor domain reveals a dramatically opened ATP-binding pocket, as if ready to exchange its bound ADP for Mg·ATP. In this state, the central β-sheet is twisted ~10° beyond the maximal amount observed in other kinesins. This configuration has only been seen in the nucleotide-free states of myosins—known as the “rigor-like” state. Fitting of this atomic model to electron density maps from cryo-electron microscopy indicates a distinct binding configuration of the motor domain to microtubules. We postulate that these properties of KIF14 are well suited for stabilizing midbody microtubules during cytokinesis.
    Graphical abstract




    Categories: Journal Articles
  • Structural Basis for the Specific Recognition of the Major Antigenic Peptide from the Japanese Cedar Pollen Allergen Cry j 1 by HLA-DP5
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Seisuke Kusano , Mutsuko Kukimoto-Niino , Yoko Satta , Noboru Ohsawa , Tomomi Uchikubo-Kamo , Motoaki Wakiyama , Mariko Ikeda , Takaho Terada , Ken Yamamoto , Yasuharu Nishimura , Mikako Shirouzu , Takehiko Sasazuki , Shigeyuki Yokoyama

    The major allergen, Cry j 1, was isolated from Japanese cedar Cryptomeria japonica (Cry j) pollen and was shown to react with immunoglobulin E antibodies in the sera from pollinosis patients. We previously reported that the frequency of HLA-DP5 was significantly higher in pollinosis patients and the immunodominant peptides from Cry j 1 bound to HLA-DP5 to activate Th2 cells. In the present study, we determined the crystal structure of the HLA-DP5 heterodimer in complex with a Cry j 1-derived nine-residue peptide, at 2.4Å resolution. The peptide-binding groove recognizes the minimal peptide with 10 hydrogen bonds, including those between the negatively charged P1 pocket and the Lys side chain at the first position in the peptide sequence. We confirmed that HLA-DP5 exhibits the same Cry j 1-binding mode in solution, through pull-down experiments using structure-based mutations of Cry j 1. We also identified the characteristic residues of HLA-DP5 that are responsible for the distinct properties of the groove, by comparing the structure of HLA-DP5 and the previously reported structures of HLA-DP2 in complexes with pDRA of the self-antigen. The comparison revealed that the HLA-DP5·pCry j 1 complex forms several hydrogen bond/salt bridge networks between the receptor and the antigen that were not observed in the HLA-DP2·pDRA complex. Evolutionary considerations have led us to conclude that HLA-DP5 and HLA-DP2 represent two major groups of the HLA-DP family, in which the properties of the P1 and P4 pockets have evolved and acquired the present ranges of epitope peptide-binding specificities.
    Graphical abstract




    Categories: Journal Articles
  • Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Inchul Shin , Kitae Han , Sangkee Rhee

    In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.
    Graphical abstract




    Categories: Journal Articles
  • Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Jo E. Lomax , Christopher M. Bianchetti , Aram Chang , George N. Phillips Jr. , Brian G. Fox , Ronald T. Raines

    Ribonuclease inhibitor (RI) is a conserved protein of the mammalian cytosol. RI binds with high affinity to diverse secretory ribonucleases (RNases) and inhibits their enzymatic activity. Although secretory RNases are found in all vertebrates, the existence of a non-mammalian RI has been uncertain. Here, we report on the identification and characterization of RI homologs from chicken and anole lizard. These proteins bind to RNases from multiple species but exhibit much greater affinity for their cognate RNases than for mammalian RNases. To reveal the basis for this differential affinity, we determined the crystal structure of mouse, bovine, and chicken RI·RNase complexes to a resolution of 2.20, 2.21, and 1.92Å, respectively. A combination of structural, computational, and bioinformatic analyses enabled the identification of two residues that appear to contribute to the differential affinity for RNases. We also found marked differences in oxidative instability between mammalian and non-mammalian RIs, indicating evolution toward greater oxygen sensitivity in RIs from mammalian species. Taken together, our results illuminate the structural and functional evolution of RI, along with its dynamic role in vertebrate biology.
    Graphical abstract




    Categories: Journal Articles
  • Crystal Structures of Ricin Toxin's Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-Neutralizing Single-Chain Antibodies
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Michael J. Rudolph , David J. Vance , Jonah Cheung , Matthew C. Franklin , Fiana Burshteyn , Michael S. Cassidy , Ebony N. Gary , Cristina Herrera , Charles B. Shoemaker , Nicholas J. Mantis

    Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines.
    Graphical abstract




    Categories: Journal Articles
  • A Conserved Noncoding Sequence Can Function as a Spermatocyte-Specific Enhancer and a Bidirectional Promoter for a Ubiquitously Expressed Gene and a Testis-Specific Long Noncoding RNA
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Misuzu Kurihara , Akira Shiraishi , Honoo Satake , Atsushi P. Kimura

    Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1 could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes. When we integrated the 6.9-kb 5′-flanking sequence of Tcam1 with or without a deletion of CNS1 linked to the enhanced green fluorescent protein gene into the chromatin of GC-2spd(ts) cells, CNS1 significantly enhanced Tcam1 promoter activity. These results indicate that CNS1 could function as a spermatocyte-specific enhancer. Interestingly, CNS1 also showed high bidirectional promoter activity in the reporter assay, and consistent with this, the Smarcd2 gene and lncRNA, designated lncRNA-Tcam1, were transcribed from adjacent regions of CNS1. While Smarcd2 was ubiquitously expressed, lncRNA-Tcam1 expression was restricted to testicular germ cells, although this lncRNA did not participate in Tcam1 activation. Ubiquitous Smarcd2 expression was correlated to CpG hypo-methylation of CNS1 and partially controlled by Sp1. However, for lncRNA-Tcam1 transcription, the strong association with histone acetylation and histone H3K4 tri-methylation also appeared to be required. The present data suggest that CNS1 is a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of Smarcd2 and lncRNA-Tcam1.
    Graphical abstract




    Categories: Journal Articles
  • DNA-recognition by a σ54 transcriptional activator from Aquifex aeolicus
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Molecular Biology

    Author(s): Natasha K. Vidangos , Johanna Heideker , Artem Lyubimov , Meindert Lamers , Yixin Huo , Jeffrey G. Pelton , Jimmy Ton , Jay Gralla , James Berger , David E. Wemmer

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4 binding sites were identified upstream (–145 and –85 base pairs) from the start of the lpxC gene, which is responsible for the first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the –145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis.
    Graphical abstract




    Categories: Journal Articles
  • Bacterial voltage-gated sodium channels (BacNaVs) from the soil, sea, and salt lakes enlighten molecular mechanisms of electrical signaling and pharmacology in the brain and heart
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Molecular Biology

    Author(s): Jian Payandeh , Daniel L. Minor Jr.

    Voltage-gated sodium channels (NaVs) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60years, functional studies of NaVs have occupied a central place in physiological and biophysical investigation of the molecular basis of excitability. Recently, structural studies of members of a large family of bacterial voltage-gated sodium channels (BacNaVs) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic NaVs have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNaVs as templates for drug development efforts.
    Graphical abstract




    Categories: Journal Articles
  • A Structural Portrait of the PDZ Domain Family
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Molecular Biology

    Author(s): Andreas Ernst , Brent A. Appleton , Ylva Ivarsson , Yingnan Zhang , David Gfeller , Christian Wiesmann , Sachdev S. Sidhu

    PDZ (PSD-95/Discs-large/ZO-1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signalling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side chain and main chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide to understanding the structural basis for the diverse specificities across the family.
    Graphical abstract




    Categories: Journal Articles
  • The PHD finger of p300 influences its ability to acetylate histone and non-histone targets
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Molecular Biology

    Author(s): Johannes G.M. Rack , Timo Lutter , Gro E.K. Bjerga , Corina Guder , Christine Ehrhardt , Signe Värv , Mathias Ziegler , Rein Aasland

    In enzymes that regulate chromatin structure, the combinatorial occurrence of modules that alter and recognise histone modifications is a recurrent feature. In this study, we explored the functional relationship between the acetyltransferase domain and the adjacent bromodomain/PHD finger region of the transcriptional coactivator p300. We found that the bromo/PHD region of p300 can bind to the acetylated catalytic domain in vitro and augment the catalytic activity of the enzyme. Deletion of the PHD finger, but not the bromodomain, impaired the ability of the enzyme to acetylate histones in vivo, while it enhanced p300 self-acetylation. A Rubinstein-Taybi syndrom-related point mutation in the p300 PHD finger resulted in increased self-acetylation, but retained the ability to acetylate histones. Hence, the PHD finger appears to negatively regulate self-acetylation. Furthermore, our data suggest that the PHD finger has a role in the recruitment of p300 to chromatin.
    Graphical abstract




    Categories: Journal Articles
  • The Enigmatic Cytoplasmic Regions of KCNH Channels
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Molecular Biology

    Author(s): João H. Morais-Cabral , Gail A. Robertson

    KCNH channels are expressed across a vast phylogenetic and evolutionary spectrum. In humans they function in a wide range of tissues and serve as biomarkers and targets for diseases such as cancer and cardiac arrhythmias. These channels share a general architecture with other voltage-gated ion channels but are distinguished by the presence of an N-terminal Per-Arnt-Sim (PAS) domain and a C-terminal domain with homology to cyclic nucleotide binding domains (referred to as the CNBh domain). Cytosolic regions outside these domains show little conservation between KCNH families but within a family are strongly conserved across species, likely reflecting variability that confers specificity to individual channel types. PAS and CNBh domains participate in channel gating, but at least twice in evolutionary history the PAS domain has been lost, and in one family it is omitted by alternate transcription to create a distinct channel subunit. In this focused review we present current knowledge of the structure and function of these cytosolic regions, discuss their evolution as modular domains, and provide our perspective on the important questions moving forward.
    Graphical abstract




    Categories: Journal Articles
  • Molecular architecture of photoreceptor phosphodiesterase elucidated by chemical cross-linking and integrative modeling
    [Aug 2014]

    Publication date: Available online 19 August 2014
    Source:Journal of Molecular Biology

    Author(s): Xiaohui Zeng-Elmore , Xiong-Zhuo Gao , Riccardo Pellarin , Dina Schneidman-Duhovny , Xiu-Jun Zhang , Katie A. Kozacka , Yang Tang , Andrej Sali , Robert J. Chalkley , Rick H. Cote , Feixia Chu

    Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery and adaptation of visual detection. Although major steps in the PDE6 activation/deactivation pathway have been identified, mechanistic understanding of PDE6 regulation is limited by the lack of knowledge about the molecular organization of the PDE6 holoenzyme (αβγγ). Here, we characterize the PDE6 holoenzyme by integrative structural determination of the PDE6 catalytic dimer (αβ), based primarily on chemical cross-linking and mass spectrometric analysis. Our models built from the high-density cross-linking data elucidate a parallel organization of the two catalytic subunits, with juxtaposed α-helical segments within the tandem regulatory GAF domains to provide multiple sites for dimerization. The two catalytic domains exist in an open configuration when compared to the structure of PDE2 in the apo state. Detailed structural elements for a differential binding of the γ-subunit to the GAFa domains of the α- and β-subunit are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.
    Graphical abstract




    Categories: Journal Articles
  • Coevolution of specificity determinants in eukaryotic glutamyl- and glutaminyl-tRNA synthetases
    [Aug 2014]

    Publication date: Available online 19 August 2014
    Source:Journal of Molecular Biology

    Author(s): Andrew Hadd , John J. Perona

    The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNAGln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNAGln and tRNAGlu with glutamate. This ancient GluRS also separately differentiated to exclude tRNAGln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNAGln and tRNAGlu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model, and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p-GluRS complex upon the diauxic shift to respiratory conditions.
    Graphical abstract




    Categories: Journal Articles
  • Functional Suppression of HAMP Domain Signaling Defects in the E. coli Serine Chemoreceptor
    [Aug 2014]

    Publication date: Available online 15 August 2014
    Source:Journal of Molecular Biology

    Author(s): Run-Zhi Lai , John S. Parkinson

    HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric E. coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA, bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: Unmodified E residues promote kinase-off output; methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer three of the HAMP bundle and followed in vivo CheA activity with a FRET-based assay. We found that an alanine or serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells, but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E, S255A) shifted Tsr output toward the kinase-off state, but two others (S255G, L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations.
    Graphical abstract




    Categories: Journal Articles
  • Ryanodine Receptors: Allosteric Ion Channel Giants
    [Aug 2014]

    Publication date: Available online 15 August 2014
    Source:Journal of Molecular Biology

    Author(s): Filip Van Petegem

    The endoplasmic (ER) and sarcoplasmic reticulum (SR) form major intracellular Ca2+ stores. Ryanodine Receptors (RyRs) are large tetrameric ion channels in the SR and ER membranes that can release the Ca2+ upon triggering. With molecular weights exceeding 2.2 MDa, they represent the pinnacle of ion channel complexity. RyRs have adopted long-range allosteric mechanisms, with pore opening resulting in conformational changes over 200Å away. Together with the tens of protein and small molecule modulators, RyRs have adopted rich and complex regulatory mechanisms. Structurally related to inositol-1,4,5-trisphosphate receptors (IP3Rs), RyRs have been studied extensively using cryo-electron microscopy. Along with more recent X-ray crystallographic analyses of individual domains, these have resulted in pseudo-atomic models. Over 500 mutations in RyRs have been linked to severe genetic disorders, which underscore their role in the contraction of cardiac and skeletal muscle. Most of these have been linked to gain-of-function phenotypes, resulting in premature or prolonged leak of Ca2+ in the cytosol. This review outlines our current knowledge on the structure of RyRs at high and low resolution, their relationship to IP3Rs, an overview of the most commonly studied regulatory mechanisms, and models that relate disease-causing mutations to altered channel function.
    Graphical abstract




    Categories: Journal Articles
  • DNA Looping Provides for “Intersegmental Hopping” by Proteins: A Mechanism for Long-Range Site Localization
    [Aug 2014]

    Publication date: Available online 15 August 2014
    Source:Journal of Molecular Biology

    Author(s): Adam J. Pollak , Aaron T. Chin , Frank L.H. Brown , Norbert O. Reich

    Studies of how transcription factors and DNA modifying enzymes passively locate specific sites on DNA have yet to be reconciled with a sufficient set of mechanisms that can adequately account for the efficiency and speed of this process. This is especially true when considering that these DNA binding/modifying proteins have diverse levels of both cellular copy numbers and genomic recognition site densities. The monomeric bacterial DNA adenine methyltransferase (Dam) is responsible for the rapid methylation of the entire chromosome (with only ~100 Dam copies per cell) and the regulated methylation of closely spaced sites which controls the expression of virulence genes in several human pathogens. Provocatively, we find Dam travels between its recognition sites most efficiently when those sites are ~500 base pairs apart. We propose that this is manifested by Dam moving between distal regions on the same DNA molecule, which is mediated by DNA looping, a phenomenon we designate as intersegmental hopping. Importantly, an intermediate found in other systems including two simultaneously bound, looped DNA strands is not involved here. Our results suggest that intersegmental hopping contributes to enzymatic processivity (multiple modifications), invoking recent reports that demonstrate DNA looping can assist in site finding. Intersegmental hopping is possibly used by other sequence specific DNA binding proteins, such as transcription factors and regulatory proteins, given certain biological context. While a general form of this mechanism is proposed by many research groups, our consideration of DNA looping in the context of processive catalysis provides new mechanistic insights and distinctions.
    Graphical abstract




    Categories: Journal Articles
  • Chromatin regulation of DNA damage repair and genome integrity in the central nervous system
    [Aug 2014]

    Publication date: Available online 14 August 2014
    Source:Journal of Molecular Biology

    Author(s): Ling Pan , Jay Penney , Li-Huei Tsai

    With the continued extension of lifespan, aging and age related diseases have become a major medical challenge to our society. Aging is accompanied by changes in multiple systems. Among these, the aging process in the central nervous system is critically important but very poorly understood. Neurons, as post-mitotic cells, are devoid of replicative associated aging processes, such as senescence and telomere shortening. However, because of the inability to self-replenish, neurons have to withstand challenge from numerous stressors over their lifetime. Many of these stressors can lead to damage of the neurons’ DNA. When the accumulation of DNA damage exceeds a neuron’s capacity for repair, or when there are deficiencies in DNA repair machinery, genome instability can manifest. The increased mutation load associated with genome instability can lead to neuronal dysfunction and ultimately to neuron degeneration. In this review, we first briefly introduce the sources and types of DNA damage and the relevant repair pathways in nervous system (summarized in Fig. 1). We then discuss the chromatin regulation of these processes and summarize our understanding of the contribution of genomic instability to neurodegenerative diseases.
    Graphical abstract




    Categories: Journal Articles