Showing papers in "Annual Review of Biochemistry in 2002"

DNA replication in eukaryotic cells

Abstract: ▪ Abstract The maintenance of the eukaryotic genome requires precisely coordinated replication of the entire genome each time a cell divides. To achieve this coordination, eukaryotic cells use an ordered series of steps to form several key protein assemblies at origins of replication. Recent studies have identified many of the protein components of these complexes and the time during the cell cycle they assemble at the origin. Interestingly, despite distinct differences in origin structure, the identity and order of assembly of eukaryotic replication factors is highly conserved across all species. This review describes our current understanding of these events and how they are coordinated with cell cycle progression. We focus on bringing together the results from different organisms to provide a coherent model of the events of initiation. We emphasize recent progress in determining the function of the different replication factors once they have been assembled at the origin.

Mammalian ABC Transporters in Health and Disease

Abstract: The ATP-binding cassette (ABC) transporters are a family of large proteins in membranes and are able to transport a variety of compounds through membranes against steep concentration gradients at the cost of ATP hydrolysis. The available outline of the human genome contains 48 ABC genes; 16 of these have a known function and 14 are associated with a defined human disease. Major physiological functions of ABC transporters include the transport of lipids, bile salts, toxic compounds, and peptides for antigen presentation or other purposes. We review the functions of mammalian ABC transporters, emphasizing biochemical mechanisms and genetic defects. Our overview illustrates the importance of ABC transporters in human physiology, toxicology, pharmacology, and disease. We focus on three topics: (a) ABC transporters transporting drugs (xenotoxins) and drug conjugates. (b) Mammalian secretory epithelia using ABC transporters to excrete a large number of substances, sometimes against a steep concentration gradient. Several inborn errors in liver metabolism are due to mutations in one of the genes for these pumps; these are discussed. (c) A rapidly increasing number of ABC transporters are found to play a role in lipid transport. Defects in each of these transporters are involved in human inborn or acquired diseases.

Order out of chaos: assembly of ligand binding sites in heparan sulfate.

Abstract: ▪ Abstract Virtually every cell type in metazoan organisms produces heparan sulfate. These complex polysaccharides provide docking sites for numerous protein ligands and receptors involved in diverse biological processes, including growth control, signal transduction, cell adhesion, hemostasis, and lipid metabolism. The binding sites consist of relatively small tracts of variably sulfated glucosamine and uronic acid residues in specific arrangements. Their formation occurs in a tissue-specific fashion, generated by the action of a large family of enzymes involved in nucleotide sugar metabolism, polymer formation (glycosyltransferases), and chain processing (sulfotransferases and an epimerase). New insights into the specificity and organization of the biosynthetic apparatus have emerged from genetic studies of cultured cells, nematodes, fruit flies, zebrafish, rodents, and humans. This review covers recent developments in the field and provides a resource for investigators interested in the incredible dive...

RNA Editing by Adenosine Deaminases That Act on RNA

Abstract: ▪ Abstract ADARs are RNA editing enzymes that target double-stranded regions of nuclear-encoded RNA and viral RNA. These enzymes are particularly abundant in the nervous system, where they diversify the information encoded in the genome, for example, by altering codons in mRNAs. The functions of ADARs in known substrates suggest that the enzymes serve to fine-tune and optimize many biological pathways, in ways that we are only starting to imagine. ADARs are also interesting in regard to the remarkable double-stranded structures of their substrates and how enzyme specificity is achieved with little regard to sequence. This review summarizes ongoing investigations of the enzyme family and their substrates, focusing on biological function as well as biochemical mechanism.

Biochemistry of Na,K-ATPase

Abstract: ▪ Abstract The Na,K-ATPase or sodium pump carries out the coupled extrusion and uptake of Na and K ions across the plasma membranes of cells of most higher eukaryotes. It is a member of the P-type ...

ATP-Dependent Nucleosome Remodeling

Abstract: It has been a long-standing challenge to decipher the principles that enable cells to both organize their genomes into compact chromatin and ensure that the genetic information remains accessible to regulatory factors and enzymes within the confines of the nucleus. The discovery of nucleosome remodeling activities that utilize the energy of ATP to render nucleosomal DNA accessible has been a great leap forward. In vitro, these enzymes weaken the tight wrapping of DNA around the histone octamers, thereby facilitating the sliding of histone octamers to neighboring DNA segments, their displacement to unlinked DNA, and the accumulation of patches of accessible DNA on the surface of nucleosomes. It is presumed that the collective action of these enzymes endows chromatin with dynamic properties that govern all nuclear functions dealing with chromatin as a substrate. The diverse set of ATPases that qualify as the molecular motors of the nucleosome remodeling process have a common history and are part of a superfamily. The physiological context of their remodeling action builds on the association with a wide range of other proteins to form distinct complexes for nucleosome remodeling. This review summarizes the recent progress in our understanding of the mechanisms underlying the nucleosome remodeling reaction, the targeting of remodeling machines to selected sites in chromatin, and their integration into complex regulatory schemes.

Error-prone repair DNA polymerases in prokaryotes and eukaryotes.

Abstract: DNA repair is crucial to the well-being of all organisms from unicellular life forms to humans. A rich tapestry of mechanistic studies on DNA repair has emerged thanks to the recent discovery of Y-family DNA polymerases. Many Y-family members carry out aberrant DNA synthesis-poor replication accuracy, the favored formation of non-Watson-Crick base pairs, efficient mismatch extension, and most importantly, an ability to replicate through DNA damage. This review is devoted primarily to a discussion of Y-family polymerase members that exhibit error-prone behavior. Roles for these remarkable enzymes occur in widely disparate DNA repair pathways, such as UV-induced mutagenesis, adaptive mutation, avoidance of skin cancer, and induction of somatic cell hypermutation of immunoglobulin genes. Individual polymerases engaged in multiple repair pathways pose challenging questions about their roles in targeting and trafficking. Macromolecular assemblies of replication-repair "factories" could enable a cell to handle the complex logistics governing the rapid migration and exchange of polymerases.

V(D)J Recombination: RAG Proteins, Repair Factors, and Regulation*

Abstract: V(D)J recombination is the specialized DNA rearrangement used by cells of the immune system to assemble immunoglobulin and T-cell receptor genes from the preexisting gene segments. Because there is a large choice of segments to join, this process accounts for much of the diversity of the immune response. Recombination is initiated by the lymphoid-specific RAG1 and RAG2 proteins, which cooperate to make double-strand breaks at specific recognition sequences (recombination signal sequences, RSSs). The neighboring coding DNA is converted to a hairpin during breakage. Broken ends are then processed and joined with the help of several factors also involved in repair of radiation-damaged DNA, including the DNA-dependent protein kinase (DNA-PK) and the Ku, Artemis, DNA ligase IV, and Xrcc4 proteins, and possibly histone H2AX and the Mre11/Rad50/Nbs1 complex. There may be other factors not yet known. V(D)J recombination is strongly regulated by limiting access to RSS sites within chromatin, so that particular sites are available only in certain cell types and developmental stages. The roles of enhancers, histone acetylation, and chromatin remodeling factors in controlling accessibility are discussed. The RAG proteins are also capable of transposing RSS-ended fragments into new DNA sites. This transposition helps to explain the mechanism of RAG action and supports earlier proposals that V(D)J recombination evolved from an ancient mobile DNA element.

Eukaryotic DNA polymerases.

Abstract: ▪ Abstract Any living cell is faced with the fundamental task of keeping the genome intact in order to develop in an organized manner, to function in a complex environment, to divide at the right time, and to die when it is appropriate. To achieve this goal, an efficient machinery is required to maintain the genetic information encoded in DNA during cell division, DNA repair, DNA recombination, and the bypassing of damage in DNA. DNA polymerases (pols) α, β, γ, δ, and ϵ are the key enzymes required to maintain the integrity of the genome under all these circumstances. In the last few years the number of known pols, including terminal transferase and telomerase, has increased to at least 19. A particular pol might have more than one functional task in a cell and a particular DNA synthetic event may require more than one pol, which suggests that nature has provided various safety mechanisms. This multi-functional feature is especially valid for the variety of novel pols identified in the last three years. T...

Neuronal Ca2+/Calmodulin-Dependent Protein Kinase II: The Role of Structure and Autoregulation in Cellular Function

Abstract: ▪ Abstract Highly enriched in brain tissue and present throughout the body, Ca2+/calmodulin-dependent protein kinase II (CaMKII) is central to the coordination and execution of Ca2+ signal transduction. The substrates phosphorylated by CaMKII are implicated in homeostatic regulation of the cell, as well as in activity-dependent changes in neuronal function that appear to underlie complex cognitive and behavioral responses, including learning and memory. The architecture of CaMKII holoenzymes is unique in nature. The kinase functional domains (12 per holoenzyme) are attached by stalklike appendages to a gear-shaped core, grouped into two clusters of six. Each subunit contains a catalytic, an autoregulatory, and an association domain. Ca2+/calmodulin (CaM) binding disinhibits the autoregulatory domain, allowing autophosphorylation and complex changes in the enzyme’s sensitivity to Ca2+/CaM, including the generation of Ca2+/CaM-independent activity, CaM trapping, and CaM capping. These processes confer a typ...

Lipoprotein Receptors in the Nervous System

Abstract: The low-density-lipoprotein (LDL) receptor family is an evolutionarily ancient gene family of structurally closely related cell-surface receptors. Members of the family are involved in the cellular uptake of extracellular ligands and regulate diverse biological processes including lipid and vitamin metabolism and cell-surface protease activity. Some members of the family also participate in cellular signaling and regulate the development and functional maintenance of the nervous system. Here we review the roles of this family of multifunctional receptors in the nervous system and focus on recent advances toward the understanding of the mechanisms by which lipoprotein receptors and their ligands transmit and modulate signals in the brain.

The Bacterial RecA Protein and the Recombinational DNA Repair of Stalled Replication Forks

Abstract: ▪ Abstract The primary function of bacterial recombination systems is the nonmutagenic repair of stalled or collapsed replication forks. The RecA protein plays a central role in these repair pathways, and its biochemistry must be considered in this context. RecA protein promotes DNA strand exchange, a reaction that contributes to fork regression and DNA end invasion steps. RecA protein activities, especially formation and disassembly of its filaments, affect many additional steps. So far, Escherichia coli RecA appears to be unique among its nearly ubiquitous family of homologous proteins in that it possesses a motorlike activity that can couple the branch movement in DNA strand exchange to ATP hydrolysis. RecA is also a multifunctional protein, serving in different biochemical roles for recombinational processes, SOS induction, and mutagenic lesion bypass. New biochemical and structural information highlights both the similarities and distinctions between RecA and its homologs. Increasingly, those differe...

The la protein

Abstract: f Abstract Ubiquitous in eukaryotic cells, the La protein associates with the 3 termini of many newly synthesized small RNAs. RNAs bound by the La protein include all nascent transcripts made by RNA polymerase III as well as certain small RNAs synthesized by other RNA polymerases. Recent genetic and biochemical analyses have revealed that binding by the La protein protects the 3 ends of these RNAs from exonucleases. This La-mediated stabilization is required for the normal pathway of pre-tRNA maturation, facilitates assembly of small RNAs into functional RNA-protein complexes, and contributes to nuclear retention of certain small RNAs. Studies of mutant La proteins have given some insights into how the La protein specifically recognizes its RNA targets. However, many questions remain regarding the molecular mechanisms by which La protein binding influences multiple steps in small RNA biogenesis. This review focuses on the roles of the La protein in small RNA biogenesis and also discusses data that implicate the La protein in the translation of specific mRNAs.

Nuclear Actin and Actin-Related Proteins in Chromatin Remodeling

Abstract: ▪ Abstract The existence and function of actin in the nucleus has been hotly debated for forty years. Recently, β-actin was found to be a component of mammalian SWI/SNF-like BAF chromatin remodeling complexes and still more recently other SWI/SNF-related chromatin remodeling complexes in yeast, flies, and man. Although the function of actin in these chromatin remodeling complexes is only starting to be explored, the fact that actin is one of the most regulated proteins in the cell suggests that control of nuclear actin may be a critical regulatory point in the control of chromatin remodeling. Actin rapidly shuttles between the nucleus and the cytoplasm offering additional sites and modes of regulation. In addition, actin-related proteins (Arps) are also components of these chromatin remodeling complexes and have been implicated in transcriptional control in yeast. The observation that the BAF chromatin remodeling complex in which actin was originally identified, is also a human tumor suppressor complex ne...

Metabolism and the control of circadian rhythms

Abstract: The core apparatus that regulates circadian rhythm has been extensively studied over the past five years. A looming question remains, however, regarding how this apparatus is adjusted to maintain coordination between physiology and the changing environment. The diversity of stimuli and input pathways that gain access to the circadian clock are summarized. Cellular metabolic states could serve to link physiologic perception of the environment to the circadian oscillatory apparatus. A simple model, integrating biochemical, cellular, and physiologic data, is presented to account for the connection of cellular metabolism and circadian rhythm.

Active site tightness and substrate fit in DNA replication.

Abstract: ▪ Abstract Various physicochemical factors influence DNA replication fidelity. Since it is now known that Watson-Crick hydrogen bonds are not necessary for efficient and selective replication of a base pair by DNA polymerase enzymes, a number of alternative physical factors have been examined to explain the efficiency of these enzymes. Among these factors are minor groove hydrogen bonding, base stacking, solvation, and steric effects. We discuss the concept of active site tightness in DNA polymerases, and consider how it might influence steric (size and shape) effects of nucleotide selection in synthesis of a base pair. A high level of active site tightness is expected to lead to higher fidelity relative to proteins with looser active sites. We review the current data on what parts and dimensions of active sites are most affected by size and shape, based on data with modified nucleotides that have been examined as polymerase substrates. We also discuss recent data on nucleotide analogs displaying higher f...

Long-Distance Electron Transfer Through DNA

Abstract: DNA molecules are able to transport electrons over long distances. In most experiments the process is stimulated by the oxidation of guanines (G), which generates guanine radical cations. The electron transport through DNA occurs in a multistep hopping mechanism with all Gs as carriers of the positive charge. The rate of each individual hopping step between the Gs decreases strongly with increase of the distance. If the (A:T) bridges between the guanines are long, adenines (A) also become charge carriers. Mismatches, single strands, and G-oxidation products can drastically diminish the efficiency of the charge transport. But in triplexes and DNA/RNA duplexes, as well as in several duplex DNA/peptide complexes, the efficacy of the charge transport is less affected. The ability of DNA molecules to transport charges over long distances could provide a mechanism for ameliorating the harmfulness of damage to DNA under the conditions of oxidative stress.

Catalytic Proficiency: The Unusual Case of OMP Decarboxylase

Abstract: ▪ Abstract Enzymes are called upon to differ greatly in the difficulty of the tasks that they perform. The catalytic proficiency of an enzyme can be evaluated by comparing the second-order rate constant (kcat/Km) with the rate of the spontaneous reaction in neutral solution in the absence of a catalyst. The proficiencies of enzymes, measured in this way, are matched by their affinity constants for the altered substrate in the transition state. These values vary from approximately ∼109 M−1 for carbonic anhydrase to ∼1023 M−1 for yeast orotidine 5′-phosphate decarboxylase (ODCase). ODCase turns its substrate over with a half-time of 18 ms, in a reaction that proceeds in its absence with a half-time of 78 million years in neutral solution. ODCase differs from other decarboxylases in that its catalytic activity does not depend on the presence of metals or other cofactors, or on the formation of a covalent bond to the substrate. Several mechanisms of transition state stabilization are considered in terms of OD...

Biological roles of proteases in parasitic protozoa.

Abstract: Proteases from a variety of protozoan parasites have been characterized at the molecular and cellular levels, and the many roles that proteases play in these organisms are coming into focus. Central roles have been proposed for proteases in diverse processes such as host cell invasion and egress, encystation, excystation, catabolism of host proteins, differentiation, cell cycle progression, cytoadherence, and both stimulation and evasion of host immune responses. Detailed structural and functional characterization of parasite proteases has led to novel insights into the workings of these fascinating catalytic machines. The possibility of developing selective inhibitors of key proteases of pathogenic parasites into novel chemotherapeutic strategies is being vigorously explored.

Great metalloclusters in enzymology.

Abstract: Metallocluster-containing enzymes catalyze some of the most basic redox transformations in the biosphere. The reactions catalyzed by these enzymes typically involve small molecules such as N2, CO, and H2 that are used to generate both chemical building blocks and energy for metabolic purposes. During the past decade, structures have been established for the iron-sulfur-based metalloclusters present in the molybdenum nitrogenase, the iron-only hydrogenase, and the nickel-carbon monoxide dehydrogenase, and for the copper-sulfide-based cluster in nitrous oxide reductase. Although these clusters are built from interactions observed in simpler metalloproteins, they contain novel features that may be relevant for their catalytic function. The mechanisms of metallocluster-containing enzymes are still poorly defined, and represent substantial and continuing challenges to biochemists, biophysicists, and synthetic chemists. These proteins also provide a window into the union of the biological and inorganic worlds that may have been relevant to the early evolution of biochemical catalysis.

The structural basis of large ribosomal subunit function

Abstract: ▪ Abstract The ribosome crystal structures published in the past two years have revolutionized our understanding of ribonucleoprotein structure, and more specifically, the structural basis of the peptide bonding forming activity of the ribosome. This review concentrates on the crystallographic developments that made it possible to solve these structures. It also discusses the information obtained from these structures about the three-dimensional architecture of the large ribosomal subunit, the mechanism by which it facilitates peptide bond formation, and the way antibiotics inhibit large subunit function. The work reviewed, taken as a whole, proves beyond doubt that the ribosome is an RNA enzyme, as had long been surmised on the basis of less conclusive evidence.

Homogeneous glycopeptides and glycoproteins for biological investigation.

Abstract: ▪ Abstract Protein glycosylation is widely recognized as a modulator of protein structure, localization, and cell-cell recognition in multicellular systems. Glycoproteins are typically expressed as mixtures of glycoforms, their oligosaccharides being generated by a template-independent biosynthetic process. Investigation of their function has been greatly assisted by sources of homogeneous material. This review summarizes current efforts to obtain homogeneous glycopeptide and glycoprotein materials by a variety of methods that draw from the techniques of recombinant expression, chemical synthesis, enzymatic transformation, and chemoselective ligation. Some of these techniques remove obstacles to glycoprotein synthesis by installing nonnative linkages and other modifications for facilitated assembly. The end purpose of the described approaches is the production of glycosylated materials for experiments relevant to the biological investigation of glycoproteins, although the strategies presented apply to oth...

Formation of unusual sugars: mechanistic studies and biosynthetic applications.

Abstract: Carbohydrates are highly abundant biomolecules found extensively in nature. Besides playing important roles in energy storage and supply, they often serve as essential biosynthetic precursors or structural elements needed to sustain all forms of life. A number of unusual sugars that have certain hydroxyl groups replaced by a hydrogen, an amino group, or an alkyl side chain play crucial roles in determining the biological activity of the parent natural products in bacterial lipopolysaccharides or secondary metabolite antibiotics. Recent investigation of the biosynthesis of these monosaccharides has led to the identification of the gene clusters whose protein products facilitate the unusual sugar formation from the ubiquitous NDP-glucose precursors. This review summarizes the mechanistic studies of a few enzymes crucial to the biosynthesis of C-2, C-3, C-4, and C-6 deoxysugars, the characterization and mutagenesis of nucleotidyl transferases that can recognize and couple structural analogs of their natural substrates and the identification of glycosyltransferases with promiscuous substrate specificity. Information gleaned from these studies has allowed pathway engineering, resulting in the creation of new macrolides with unnatural deoxysugar moieties for biological activity screening. This represents a significant progress toward our goal of searching for more potent agents against infectious diseases and malignant tumors.

Eukaryotic ribonuclease P: a plurality of ribonucleoprotein enzymes.

Abstract: ▪ Abstract Ribonuclease P (RNase P) is an essential endonuclease that acts early in the tRNA biogenesis pathway. This enzyme catalyzes cleavage of the leader sequence of precursor tRNAs (pre-tRNAs), generating the mature 5′ end of tRNAs. RNase P activities have been identified in Bacteria, Archaea, and Eucarya, as well as organelles. Most forms of RNase P are ribonucleoproteins, i.e., they consist of an essential RNA subunit and protein subunits, although the composition of the enzyme in mitochondria and chloroplasts is still under debate. The recent purification of the eukaryotic nuclear RNase P has demonstrated a significantly larger protein content compared to the bacterial enzyme. Moreover, emerging evidence suggests that the eukaryotic RNase P has evolved into at least two related nuclear enzymes with distinct functions, RNase P and RNase MRP. Here we review current information on RNase P, with emphasis on the composition, structure, and functions of the eukaryotic nuclear holoenzyme, and its relatio...

Catalytic Strategies of the Hepatitis Delta Virus Ribozymes

Abstract: The hepatitis delta virus (HDV) ribozymes are self-cleaving RNA sequences critical to the replication of a small RNA genome. A recently determined crystal structure together with biochemical and biophysical studies provides new insight into the possible catalytic mechanism of these ribozymes. The HDV ribozymes are examples of naturally occurring small ribozymes that catalyze cleavage of the RNA backbone with a rate enhancement of 10(6)- to 10(7)-fold over the uncatalyzed rate. To achieve this level of rate enhancement, the HDV ribozymes have been proposed to employ several catalytic strategies that include the use of metal ions, intrinsic binding energy, and a novel example of general acid-base catalysis with a cytosine side chain acting as a proton donor or acceptor.

Mechanisms of Fast Protein Folding

Abstract: ▪ Abstract An explosion of in vitro experimental data on the folding of proteins has revealed many examples of folding in the millisecond or faster timescale, often occurring in the absence of stable intermediate states. We review experimental methods for measuring fast protein folding kinetics, and then discuss various analytical models used to interpret these data. Finally, we classify general mechanisms that have been proposed to explain fast protein folding into two catagories, heterogeneous and homogeneous, reflecting the nature of the transition state. One heterogeneous mechanism, the diffusion-collision mechanism, can be used to interpret experimental data for a number of proteins.

Discoveries of Vitamin B12 and Selenium Enzymes

Abstract: My undergraduate education at Cornell University was followed by graduate studies on methane fermentations under the guidance of H.A. Barker at the University of California, Berkeley. My Ph.D. degree was granted in June 1949. Two anaerobic microorganisms isolated from the mud flats of San Francisco Bay served as sources of biochemical research material for later studies at the National Institutes of Health in Bethesda. These organisms, Methanococcus vannielii and Clostridium sticklandii, proved to be especially rich sources of selenium-dependent enzymes and seleno-tRNAs. New B12 coenzyme-dependent enzymes that catalyzed intermediate steps in the anaerobic conversion of lysine to fatty acids and ammonia were isolated from C. sticklandii and characterized. My research efforts since 1970 have dealt primarily with various aspects of selenium biochemistry. We have shown that selenium is an essential constituent of several enzymes in prokaryotes. Se is present in these either as a selenocysteine residue in the protein or alternatively, in a few molybdoenzymes, as a component of a bound cofactor. Recent studies with a human adenocarcinoma cell line led to the unexpected discovery that selenocysteine occurs in mammalian thioredoxin reductase. The selenium located in a redox center of this enzyme is essential for catalytic activity.

My career in molecular biology

Abstract: Norman Davidson's training as a physical chemist led him to make key early contributions to the chemistry of DNA. He described the details of DNA denaturation and renaturation, concepts that still form the basis for understanding hybridization. He also applied the single-molecule resolution of the electron microscope to describing the chemistry of circular DNA, mapping specific genes, and characterizing heteroduplexes. The latter became a dominant tool for the study of nucleic acids and contributed to our knowledge of transcription, polyadenylation, and retroviral structure. The advent of cDNA cloning and restriction enzymes enabled Davidson to describe the diversity of Drosophila actin genes and to isolate the gene encoding cAMP phosphodiesterase. Davidson then turned his attention to neuroscience and participated in cDNA cloning, oocyte expression, and structure-function studies of nicotinic acetylcholine receptors, voltage-gated sodium channels, a GABA transporter, a G protein-gated potassium channel, and calcium channels. His interests also extended to synaptic plasticity, and he helped to define the role of neuronal nitric oxide synthase and of trkB receptors. His final experiments concerned the role of protein kinase A in long-term potentiation. (The abstract was written posthumously by a colleague.)