Annual Review of Biochemistry - Volume 65, 1996

Multiple relationships have been noted between DNA repair and transcription in both prokaryotic and eukaryotic cells. First, in both prokaryotes and eukaryotes nucleotide excision repair of the template strand of transcriptionally active regions of the genome is faster than in the coding strand. In prokaryotes the biochemical basis for this kinetic difference appears to be related to the specific coupling of repair to arrested transcription by RNA polymerase. The biochemical basis for strand-specific repair in eukaryotes is unknown. Second, in eukaryotes some or all of the subunits of transcription factor IIH (TFIIH) are required for nucleotide excision repair. The biological significance of this dual function of TFIIH proteins is not obvious. Finally, there are indications that the genes CSA and CSB, which are implicated in the human hereditary disease Cockayne syndrome, may have a role in transcription.

In nucleotide excision repair DNA damage is removed through incision of the damaged strand on both sides of the lesion, followed by repair synthesis, which fills the gap using the intact strand as a template, and finally ligation. In prokaryotes the damaged base is removed in a 12-13 nucleotide (nt)-long oligomer; in eukaryotes including humans the damage is excised in a 24-32 nt-long fragment. Excision in Escherichia coli is accomplished by three proteins designated UvrA, UvrB, and UvrC. In humans, by contrast, 16 polypeptides including seven xeroderma pigmentosum (XP) proteins, the trimric replication protein A [RPA, human single-stranded DNA binding protein (HSSB)], and the multisubunit (7-10) general transcription factor TFIIH are required for the dual incisions. Transcribed strands are specifically targeted for excision repair by a transcription-repair coupling factor both in E. coli and in humans. In humans, excision repair is an important defense mechanism against the two major carcinogens, sunlight and cigarette smoke. Individuals defective in excision repair exhibit a high incidence of cancer while individuals with a defect in coupling transcription to repair suffer from neurological and skeletal abnormalities.

Selenocysteine is recognized as the 21st amino acid in ribosome-mediated protein synthesis and its specific incorporation is directed by the UGA codon. Unique tRNAs that have complementary UCA anticodons are aminoacylated with serine, the seryl-tRNA is converted to selenocysteyl-tRNA and the latter binds specifically to a special elongation factor and is delivered to the ribosome. Recognition elements within the mRNAs are essential for translation of UGA as selenocysteine. A reactive oxygen-labile compound, selenophosphate, is the selenium donor required for synthesis of selenocysteyl-tRNA. Selenophosphate synthetase, which forms selenophosphate from selenide and ATP, is found in various prokaryotes, eukaryotes, and archaebacteria. The distribution and properties of selenocysteine-containing enzymes and proteins that have been discovered to date are discussed. Artificial selenoenzymes such as selenosubtilisin have been produced by chemical modification. Genetic engineering techniques also have been used to replace cysteine residues in proteins with selenocysteine. The mechanistic roles of selenocysteine residues in the glutathione peroxidase family of enzymes, the 5′ deiodinases, formate dehydrogenases, glycine reductase, and a few hydrogenases are discussed. In some cases a marked decrease in catalytic activity of an enzyme is observed when a selenocysteine residue is replaced with cysteine. This substitution caused complete loss of glycine reductase selenoprotein A activity.

Mismatch repair stabilizes the cellular genome by correcting DNA replication errors and by blocking recombination events between divergent DNA sequences. The reaction responsible for strand-specific correction of mispaired bases has been highly conserved during evolution, and homologs of bacterial MutS and MutL, which play key roles in mismatch recognition and initiation of repair, have been identified in yeast and mammalian cells. Inactivation of genes encoding these activities results in a large increase in spontaneous mutability, and in the case of mice and men, predisposition to tumor development.

Eukaryotic cells have multiple mechanisms for repairing damaged DNA. 0⁶-methylguanine-DNA methyltransferase directly reverses some simple alkylation adducts. However, most repair strategies excise lesions from DNA. Two major pathways are base excision repair (BER), which eliminates single damaged-base residues, and nucleotide excision repair (NER), which excises damage within oligomers that are 25-32 nucleotides long. The specialized DNA glycosylases and AP endonucleases of BER act on spontaneous and induced DNA alterations caused by hydrolysis, oxygen free radicals, and simple alkylating agents. NER utilizes many proteins (including the XP proteins in humans) to remove the major UV-induced photoproducts from DNA, as well as other types of modified nucleotides. Different DNA polymerases and ligases are used to complete the separate pathways. Some organisms have alternative schemes, which include the use of photolyases and a specific UV-endonuclease for repairing UV damage to DNA. Finally, double-strand breaks in DNA are repaired by mechanisms that involve recombination proteins and, in mammalian cells, a DNA protein kinase.

DNA helicases are essential motor proteins that function to unwind duplex DNA to yield the transient single-stranded DNA intermediates required for replication, recombination, and repair. These enzymes unwind duplex DNA and translocate along DNA in reactions that are coupled to the binding and hydrolysis of 5′-nucleoside triphosphates (NTP). Although these enzymes are essential for DNA metabolism, the molecular details of their mechanisms are only beginning to emerge. This review discusses mechanistic aspects of helicasecatalyzed DNA unwinding and translocation with a focus on energetic (thermodynamic), kinetic, and structural studies of the few DNA helicases for which such information is available. Recent studies of DNA and NTP binding and DNA unwinding by the Escherichia coli (E. coli) Rep helicase suggest that the Rep helicase dimer unwinds DNA by an active, rolling mechanism. In fact, DNA helicases appear to be generally oligomeric (usually dimers or hexamers), which provides the helicase with multiple DNA binding sites. The apparent mechanistic similarities and differences among these DNA helicases are discussed.

In spite of forty years of effective chemotherapy for tuberculosis, the molecular mechanisms of antibacterial compounds in Mycobacterium tuberculosis have only recently been revealed. Broad spectrum antibacterials, including streptomycin, rifampicin, and fluoroquinolones have been demonstrated to act on the same targets in M. tuberculosis as they do in E. coli. Resistance to these agents results from single mutagenic events that lead to amino acid substitutions in their target proteins. The mechanisms of action of the unique antitubercular drugs, including isoniazid, ethambutol, and pyrazinamide have also recently been defined. Resistance to isoniazid can be caused either by mutations in the katG-encoded catalase-peroxidase, the enzyme responsible for drug activation, or by the molecular target, the inhA-encoded long chain enoyl-ACP reductase. Ethambutol appears to block specifically the biosynthesis of the arabinogalactan component of the mycobacterial cell envelope, and pyrazinamide has no known target. With the resurgence of tuberculosis and the appearance of strains which are multiply resistant to the above compounds, present tuberculosis chemotherapies are threatened. New approaches to the treatment of multi drug-resistant tuberculosis are needed.

Prenylation is a class of lipid modification involving covalent addition of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. Known prenylated proteins include fungal mating factors, nuclear lamins, Ras and Ras-related GTP-binding proteins (G proteins), the subunits of trimeric G proteins, protein kinases, and at least one viral protein. Prenylation promotes membrane interactions of most of these proteins, which is not surprising given the hydrophobicity of the lipids involved. In addition, however, prenylation appears to play a major role in several protein-protein interactions involving these species. The emphasis in this review is on the enzymology of prenyl protein processing and the functional significance of prenylation in cellular events. Several other recent reviews provide more detailed coverage of aspects of prenylation that receive limited attention here owing to length restrictions (1-4).

Protein transport across the endoplasmic reticulum membrane can occur by two pathways, a co- and a post-translational one. In both cases, polypeptides are first targeted to translocation sites in the membrane by virtue of their signal sequences and then transported across or inserted into the phospholipid bilayer, most likely through a protein-conducting channel. Key components of the translocation apparatus have now been identified and the translocation pathways Seem likely to be related to each other but mechanistically distinct. Protein transport across the bacterial inner membrane is both similar to and different from the process in eukaryotes. Other pathways of protein translocation exist that bypass the ones involving classical signal sequences.

Recently a number of α-amino acid transport proteins and corresponding cDNA clones have been isolated and categorized into gene families. The “CAT family” contains two members that mediate high-affinity Na⁺-independent transport of cationic amino acids in many tissues, and a third member that encodes a liver-specific low-affinity activity. The “glutamate transporter family” contains at least four members that mediate Na⁺-dependent glutamate/aspartate uptake and two members that are selective for neutral amino acids. The glutamate transporters are expressed at high levels in both glia and neurons of the central nervous system. The Na⁺/Cl^--dependent proline transporter (PROT) belongs to a large superfamily of neurotransmitter transporters and is expressed in regions of the brain that contain glutamanergic neurons. All four glycine transporters of the “GLYT family” also belong to the neurotransmitter superfamily and exhibit the greatest expression in the central nervous system. The “rBAT/4F2hc family” of proteins induce both neutral and cationic amino acid uptake when expressed in Xenopus oocytes. Cystinuria is linked to specific mutations in the rBAT sequence.

Telomeres are the components of chromosome ends that provide stability and allow the complete replication of the ends. Telomere length is maintained by a balance between processes that lengthen and those that shorten telomeres. Telomeraseis a ribonucleoprotein polymerase that specifically elongates telomeres. In human cells telomere length is not maintained and telomerase is not active in some tissues. In tumors, however, telomerase is active and may be required for the growth of cancer cells. Thus understanding telomerase and telomere length regulation may help us understand tumor progression. Evidence from various organisms suggests that several factors influence telomere length regulation, such as telomere binding proteins, telomere capping proteins, telomerase, and DNA replication enzymes. Understanding how these factors interact to coordinate the regulation of telomere length will allow a more complete understanding of telomere function in the cell.

Intervening sequences are removed from nuclear pre-mRNAs in a well-defined multi-step pathway. Small nuclear ribonucleoprotein particles (snRNPs) and numerous protein factors are essential for the formation of the active spliceosome in which intron excision proceeds in two successive transesterification reactions. Important elements for catalysis are the RNA moieties of the snRNPs that align the pre-mRNA splice sites in the active center of the spliceosome. Although pre-mRNA splicing is almost certainly RNA-mediated, both snRNA-associated proteins and non-snRNP splicing factors participate in each step of the splicing reaction. Splicing proteins exert auxiliary functions in the recognition, selection, and juxtaposition of the splice sites and drive conformational changes during spliceosome assembly and catalysis. Many splicing factors have been isolated in recent years and corresponding cDNAs have been cloned. This review summarizes the structure and function of mammalian proteins which are essential components of the constitutive splicing machinery.

In conditions of starvation, the free living amoebae of Dictyostelium enter a developmental program: The cells aggregate by chemotaxis to form a multicellular structure that undergoes morphogenesis and cell-type differentiation. These processes are mediated by a family of cell surface CAMP receptors (CARS) that act on a specific heterotrimeric G protein to stimulate actin polymerization, activation of adenylyl and guanylyl cyclases, and a host of other responses. Most of the components in these pathways have mammalian counterparts. The accessible genetics of this unicellular organism facilitate structure-function analysis and enable the discovery of novel genes involved in the regulation of these important pathways.

Lectins are responsible for cell surface sugar recognition in bacteria, animals, and plants. Examples include bacterial toxins; animal receptors that mediate cellcell interactions, uptake of glycoconjugates, and pathogen neutralization; and plant toxins and mitogens. The structural basis for selective sugar recognition by members of all of these groups has been investigated by x-ray crystallography. Mechanisms for sugar recognition have evolved independently in diverse protein structural frameworks, but share some key features. Relatively low affinity binding sites for monosaccharides are formed at shallow indentations on protein surfaces. Selectivity is achieved through a combination of hydrogen bonding to the sugar hydroxyl groups with van der Waals packing, often including packing of a hydrophobic sugar face against aromatic amino acid side chains. Higher selectivity of binding is achieved by extending binding sites through additional direct and water-mediated contacts between oligosaccharides and the protein surface. Dramatically increased affinity for oligosaccharides results from clustering of simple binding sites in oligomers of the lectin polypeptides. The geometry of such oligomers helps to establish the ability of the lectins to distinguish surface arrays of polysaccharides in some instances and to crosslink glycoconjugates in others.

Cells in tissues share ions, second messengers, and small metabolites through clusters of intercellular channels called gap junctions. This type of intercellular communication permits coordinated cellular activity. Intercellular channels are formed from two oligomeric integral membrane protein assemblies, called connexons, which span two adjacent cells’ plasma membranes and join in a narrow, extracellular “gap.” Connexons are formed from connexins, a highly related multigene family consisting of at least 13 members. Since the cloning of the first connexin in 1986, considerable progress has been made in our understanding of the complex molecular switches that control the formation and permeability of the intercellular channels. Analysis of the mechanisms of channel assembly has revealed the selectivity of inter-connexin interactions and uncovered novel characteristics of the channel permeability and gating behavior. Structure-function studies provide a molecular understanding of the significance of connexin diversity and demonstrate the unique regulation of connexins by tyrosine kinases and oncogenes.

Rhizobia elicit on their specific leguminous hosts the formation of new organs, called nodules, in which they fix nitrogen. The rhizobia] nodulation genes specify the synthesis of lipo-chitooligosaccharide signals, the Nod factors (NFs). Each rhizobial species has a characteristic set of nodulation genes that specifies the length of the chitooligosaccharide backbone and the type of substitutions at both ends of the molecule, thus making the NFs specific for a given plant host. At extremely low concentrations, purified NFs are capable of eliciting on homologous legume hosts many of the plant developmental responses characteristic of the bacteria themselves, including cell divisions, and the triggering of a plant organogenic program. This review summarizes our current knowledge on the biosynthesis, structure, and function of this new class of signaling molecules. Finally we discuss the possibility that these signals could be part of a new family of plant lipochitooligosaccharide growth regulators.

Electron-transfer (ET) reactions are key steps in a diverse array of biological transformations ranging from photosynthesis to aerobic respiration. A powerful theoretical formalism has been developed that describes ET rates in terms of two parameters: the nuclear reorganization energy (γ) and the electroniccoupling strength (HAB). Studies of ET reactions in ruthenium-modified proteins have probed γ and HAB in several metalloproteins (cytochrome c, myoglobin, azurin). This work has shown that protein reorganization energies are sensitive to the medium surrounding the redox sites and that an aqueous environment, in particular, leads to large reorganization energies. Analyses of electronic-coupling strengths suggest that the efficiency of long-range ET depends on the protein secondary structure: β sheets appear to mediate coupling more efficiently than α-helical structures, and hydrogen bonds play a critical role in both.

This review focuses on molecular mechanisms that underlie the communication between the nuclear and mitochondrial genomes in eukaryotic cells. These genomes interact in at least two ways. First, they contribute essential subunit polypeptides to important mitochondrial proteins; second, they collaborate in the synthesis and assembly of these proteins. The first type of interaction is important for the regulation of oxidative energy production. Isoforms of the nuclear-coded subunits of cytochrome c oxidase affect the catalytic functions of its mitochondrially coded subunits. These isoforms are differentially regulated by environmental and developmental signals and probably allow tissues to adjust their energy production to different energy demands. 'Ihe second type of interaction requires the bidirectional flow of information between the nucleus and the mitochondrion. Communication from the nucleus to the mitochondrion makes use of proteins that are translated in the cytosol and imported by the mitochondrion. Communication from the mitochondrion to the nucleus involves metabolic signals and one or more signal transduction pathways that function across the inner mitochondrial membrane. An understanding of both types of interaction is important for an understanding of OXPHOS diseases and aging.

Hematopoietic hormones/cytokines and receptors regulate a wide variety of biological activities and are important in medicine. Through recent biochemical, biophysical, and structural studies we are beginning to understand how these molecules work at the molecular level. These extracellular hormones activate their transmembrane receptors by causing them to oligomerize. The receptor oligomers in turn activate intracellular tyrosine kinase molecules which then activate transcription factors (the JAK-STAT pathways). This review centers on the molecular basis for hormone-receptor binding, and how this information is useful in understanding protein-protein interactions and for the design of second generation molecules.