Spliced adenovirus-associated virus RNA.

We describe the structure of cytoplasmic RNA species transcribed from the DNA of adenovirus-associated virus, a defective parvovirus. The RNA was hybridized with minus strand template DNA and visualized in the electron microscope. Alternatively, the DNA.RNA duplex molecules were digested with nuclease S1 or Escherichia coli exonuclease VII and analyzed by agarose gel electrophoresis. A set of RNA species was observed with 5' terminal at map positions 5, 13, 19, or 39 and a 3' terminus and poly(A) tail at position 96 (one map unit is equivalent to 1% of genome length). Most of these RNAs are spliced and lack sequences approximately between positions 40 and 49. Some RNA preparations also contained unspliced molecules with 5' and 3' terminal at positions similar to those in the spliced RNA.     

Cocrystal structure of an editing complex of Klenow fragment with DNA.

High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.     

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP[alpha S]) was introduced into the 3' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such "capped" 3' ends were found to be resistant to exonuclease III digestion. Moreover, the resistance to digestion is great enough that, under conditions used by us, just one strand of a double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands. When digestion with exonuclease III is limited and is followed by S1 nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this was thousands of nucleotides can be selectively removed from one end of a restriction fragment. In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and S1 nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.     

Mechanism of Action of Ribonuclease H Isolated from Avian Myeloblastosis Virus and Escherichia coli

Purified preparations of RNA-dependent DNA polymerase isolated from avain myeloblastosis virus contain RNase H activity. Labeled ribohomopolymers are degraded in the presence of their complementary deoxyribopolymer, except [(3)H]poly(U).poly(dA). The degradation products formed from [(3)H]poly(A).poly(dT) were identified as oligonucleotides containing 3'-hydroxyl and 5'-phosphate termini, while AMP was not detected. The nuclease has been characterized as a processive exonuclease that requires ends of poly(A) chains for activity. Exonucleolytic attack occurs in both 5' to 3' and 3' to 5' directions.RNase H has also been purified from E. coli. This nuclease degrades all homoribopolymers tested in the presence of their complementary deoxyribopolymers to yield oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. E. coli RNase H has been characterized as an endonuclease.     

Repair of depurinated DNA in vitro by enzymes purified from human lymphoblasts.

Alkali-labile lesions introduced into T7 DNA by treatment with methyl methanesulfonate were removed and the DNA was repaired by incubation with DNA polymerase alpha and nuclease from a human lymphoblastoid line followed by the addition of DNA ligase. The nuclease preparation contains both apurinic endonuclease and 5'-3' exonuclease activities. Dinucleotides appear to be the first product of exonuclease action. Repair of methyl methanesulfonate-induced damage can occur by the insertion of only a few nucleotides per lesion as in vivo.     

DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies.

Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3'----5' exonuclease activity. A mutant DNA polymerase with very little (less than or equal to 1%) 3'----5' exonuclease activity was generated. In vivo, the 3'----5' exonuclease-deficient DNA polymerase produced the highest level of spontaneous mutation observed in T4, 500- to 1800-fold above that of wild type. The large reduction in 3'----5' exonuclease activity appears to be due to two amino acid substitutions: Glu-191 to Ala and Asp-324 to Gly. Protein sequence similarities have been observed between sequences in the Escherichia coli DNA polymerase I 3'----5' exonuclease domain and conserved sequences in eukaryotic, viral, and phage DNA polymerases. It has been proposed that the conserved sequences contain metal ion binding ligands that are required for 3'----5' exonuclease activity; however, we find that some proposed T4 DNA polymerase metal binding residues are not essential for 3'----5' exonuclease activity. Thus, our T4 DNA polymerase studies do not support the hypothesis by Bernad et al. [Bernad, A., Blanco, L., Lazaro, J.M., Martin, G. & Salas, M. (1989) Cell 59, 219-228] that many DNA polymerases, including T4 DNA polymerase, share an extensively conserved 3'----5' exonuclease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.     

Model for regulation of Escherichia coli DNA repair functions.

A feedback loop for the regulation of the rec/lex-mediated DNA repair system is proposed. This model was formulated from experiments on the genetic and metabolic regulation of the rate of synthesis of protein X performed in this laboratory, and from genetic data obtained in other laboratories. Protein X is proposed to prevent DNA degradation by the recBC-coded exonuclease. The model states tht: (1) The lex (or exrA in E. coli B) gene codes for a repressor. (2) This repressor binds to an operator region of DNA consisting of the tif-zab region at 51 minutes on the E. coli chromosome. (3) The operator region controls the production of several proteins involved in DNA repair, including protein X. (4) The recA gene product is required to remove the lex-coded repressor from the operator. Thre recA gene could code for an antirepressor (inducer protein or a protease) or a modifer of recBC nuclease action; (5) Low molecular weight products of DNA degradation are effectors that activate the system. (6) Protein X limits recBC nuclease action by binding to single-stranded DNA.     

A mechanistic basis for Mre11-directed DNA joining at microhomologies

Repair of DNA double-strand breaks in vertebrate cells occurs mainly by an end-joining process that often generates junctions with sequence homologies of a few nucleotides. Mre11 is critical for this mode of repair in budding yeast and has been implicated in the microhomology-based joining. Here, we show that Mre11 exonuclease activity is sensitive to the presence of heterologous DNA, and to the structure and sequence of its ends. Addition of mismatched DNA ends stimulates degradation of DNA by Mre11, whereas cohesive ends strongly inhibit it. Furthermore, if a sequence identity is revealed during the course of degradation, it causes Mre11 nuclease activity to pause, thus stabilizing the junction at a site of microhomology. A nuclease-deficient Mre11 mutant that still binds DNA can also stimulate degradation by wild-type Mre11, suggesting that Mre11-DNA complexes may interact to bridge DNA ends and facilitate DNA joining.     

Proofreading by DNA polymerase III of Escherichia coli depends on cooperative interaction of the polymerase and exonuclease subunits.

The polymerase subunit (alpha) of Escherichia coli DNA polymerase III holoenzyme and the 3'----5' exonuclease subunit (epsilon) are each less active separately than together in the holoenzyme core (an assembly of alpha, epsilon, and theta subunits). In a complex formed from purified alpha and epsilon subunits, polymerase activity increased 2-fold, and that of the 3'----5' exonuclease increased 10- to 80-fold. The alpha-epsilon complex contains one each of the subunits as does the core. Stimulation of 3'----5' exonuclease activity is due mainly to a greatly increased affinity of the epsilon subunit for the 3'-hydroxyl terminus, resulting from DNA binding by the alpha subunit. Proofreading in the course of DNA synthesis by the alpha-epsilon complex was indistinguishable from that of the core. These findings identify the participation of the alpha subunit in proofreading by polymerase III holoenzyme and suggest that the fidelity of DNA replication may be influenced by the relative levels of the alpha and epsilon subunits in the cell.     

Site-specific cleavage of duplex DNA by a semisynthetic nuclease via triple-helix formation.

A Lys-84----Cys mutant staphylococcal nuclease was selectively linked to the 5' and/or 3' terminus of a thiol-containing polypyrimidine oligonucleotide via a disulfide bond. The oligonucleotide-staphylococcal nuclease adduct is capable of binding to a homopurine-homopyrimidine region of Watson-Crick duplex DNA by the formation of a triple-helical structure. Upon the addition of Ca2+, the nuclease cleaves DNA at sites adjacent to the homopurine tract. Specific double-strand cleavage occurred predominantly at A + T-rich sites to the 5' side of the homopurine tract for both the 5'-derivatized and the 5',3'-diderivatized nucleases; the 3'-derivatized nuclease gave no cleavage. The cleavage pattern is asymmetric and consists of multiple cleavage sites shifted to the 5' side on each strand, centered at the terminal base pair of the binding site. Microgram amounts of plasmid pDP20 DNA (4433 base pairs) containing a homopurine-homopyrimidine tract were selectively cleaved by a semisynthetic nuclease with greater than 75% efficiency at room temperature within 1 hr. Cleavage reaction conditions were optimized with respect to pH, temperature, reaction times, and reaction components. Semisynthetic nucleases of this type should provide a powerful tool in chromosomal DNA manipulations.     

p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex.

Fen1 or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-DNA junctions at the 5' ends of immature Okazaki fragments prior to their ligation into a continuous DNA strand. The gene is also necessary for repair of damaged DNA in yeast. We report that human proliferating-cell nuclear antigen (PCNA) associates with human Fen1 with a Kd of 60 nM and an apparent stoichiometry of three Fen1 molecules per PCNA trimer. The Fen1-PCNA association is seen in cell extracts without overexpression of either partner and is mediated by a basic region at the C terminus of Fen1. Therefore, the polymerase delta-PCNA-Fen1 complex has all the activities associated with prokaryotic DNA polymerases involved in replication: 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Although p21, a regulatory protein induced by p53 in response to DNA damage, interacts with PCNA with a comparable Kd (10 nM) and a stoichiometry of three molecules of p21 per PCNA trimer, a p21-PCNA-Fen1 complex is not formed. This mutually exclusive interaction suggests that the conformation of a PCNA trimer switches such that it can either bind p21 or Fen1. Furthermore, overexpression of p21 can disrupt Fen1-PCNA interaction in vivo. Therefore, besides interfering with the processivity of polymerase delta-PCNA, p21 also uncouples Fen1 from the PCNA scaffold.     

The crystal structure of exonuclease RecJ bound to Mn2+ ion suggests how its characteristic motifs are involved in exonuclease activity

RecJ, a 5' to 3' exonuclease specific for single-stranded DNA, functions in DNA repair and recombination systems. We determined the crystal structure of RecJ bound to Mn(2+) ion essential for its activity. RecJ has a novel fold in which two domains are interconnected by a long helix, forming a central groove. Mn(2+) is located on the wall of the groove and is coordinated by conserved residues characteristic of a family of phosphoesterases that includes RecJ proteins. The groove is composed of residues conserved among RecJ proteins and is positively charged. These findings and the narrow width of the groove indicate that the groove binds single- instead of double-stranded DNA.     

Cellular proteins specifically bind single- and double-stranded DNA and RNA from the initiation site of a transcript that crosses the origin of DNA replication of herpes simplex virus 1

The small-component origins of herpes simplex virus 1 DNA synthesis are transcribed late in infection by an RNA with heterogeneous initiation sites approximately 290-360 base pairs from the origins. We report that cellular proteins react with a labeled RNA probe representing the 5' terminus of a subset of this RNA but not with the complementary strand of this RNA. The proteins form two complexes. Complex 2 was formed by all nuclear extracts tested, whereas complex 1 was invariably formed by proteins present only in nuclear extracts of mock-infected cells. Complex 1 protects a contiguous stretch of 40 nucleotides of the labeled RNA probe from nuclease degradation. Formation of complex 1 was competitively inhibited in a sequence-specific fashion by single-stranded RNA and DNA and by double-stranded RNA and DNA. The protein(s) forming complex 1 is, thus, quite distinct from known nucleic acid-binding proteins in that they recognize a specific nucleotide sequence, irrespective of the nature (single- and double-stranded RNA and DNA) of the nucleic acid. We conclude the following: (i) the proteins forming complex 1 and 2 are probably different, (ii) complex 1 is neither required throughout infection for viral replication nor able to hinder viral replication in cells in culture, and (iii) cells susceptible to infection encode one or more proteins that recognize specific sequences in single-stranded nucleic acids; either these proteins impart a compatible conformation on single-stranded nucleic acids with the conformation of the same strand in the double-stranded nucleic acid, or these proteins confer a specific, distinct conformation to both single-stranded and double-stranded nucleic acids.     

Contacts between DNA gyrase and its binding site on DNA: features of symmetry and asymmetry revealed by protection from nucleases.

DNA gyrase supercoils DNA by passing one DNA segment through another by means of a reversible double-strand break at specific DNA sites. We determined the nucleotide sequence of two highly preferred gyrase binding sites and analyzed the grip of gyrase on the DNA by using protection from nuclease attack. The DNA-breakage site of gyrase was centered in about 50 base pairs (bp) of DNA that was completely protected from DNase I and flanked by DNA regions cut at average intervals of 9.9 bases. The same pattern of protection from DNase I was observed with topoisomerase II', an enzyme that shares structural homology with gyrase. The gyrase site of DNA breakage was off-center in the 140 bp of DNA protected from exonuclease III digestion. ATP or inhibitors of gyrase had little specific effect on DNase I protection. On addition of a nonhydrolyzable analogue of ATP, previously stable barriers to exonuclease III were invaded and new barriers appeared. We discuss a detailed model uniting these results with previous data on gyrase structure and mechanism.     

Reduced in vivo mutagenesis by mutant herpes simplex DNA polymerase involves improved nucleotide selection.

We present evidence that mutation frequencies in a mammalian system can vary according to the replication fidelity of the DNA polymerase. We demonstrated previously that several derivatives of herpes simplex virus type 1 that encode polymerases resistant to various antiviral drugs (e.g., nucleotide analogues) also produce reduced numbers of spontaneous mutants. Here we show that the DNA polymerase from one antimutator virus exhibits enhanced replication fidelity. First, the antimutator virus showed a reduced response to known mutagens that promote base mispairing during DNA replication (N-methyl-N'-nitro-N-nitrosoguanidine, 5-bromo-deoxyuridine). Second, purified DNA polymerase from the antimutator produced fewer replication errors in vitro, based on incorporation of mispaired nucleotides or analogues with abnormal sugar rings. We have investigated possible mechanisms for the enhanced fidelity of the antimutator polymerase. We show that the mutant enzyme has altered interactions with nucleoside triphosphates, as indicated by its resistance to nucleotide analogues and elevated Km values for normal nucleoside triphosphates. We present evidence against increased proofreading by an associated 3',5' exonuclease (as seen for T4 bacteriophage antimutator polymerases), based on nuclease levels in the mutant polymerase. We propose that reduced affinity of the polymerase for nucleoside triphosphates accounts for the antimutator phenotype by accentuating differences in base-pair stability, thus facilitating selection of correct nucleotides.     

Bacterial modification of LPS and resistance to antimicrobial peptides

Antimicrobial peptides (APs) are ubiquitous in nature and are thought to kill micro-organisms by affecting membrane integrity. These positively charged peptides interact with negative charges in the LPS of Gram-negative bacteria. A common mechanism of resistance to AP killing is LPS modification. These modifications include fatty acid additions, phosphoethanolamine (PEtN) addition to the core and lipid A regions, 4-amino-4-deoxy-L-arabinose (Ara4N) addition to the core and lipid A regions, acetylation of the O-antigen, and possibly hydroxylation of fatty acids. In Salmonella typhimurium, LPS modifications are induced within host tissues by the two-component regulatory systems PhoPQ and PmrAB. PmrAB activation results in AP resistance by Ara4N addition to lipid A through the activation of at least 8 genes, 7 of which are transcribed as an operon. Loss of this operon and, therefore, Ara4N LPS modification, affects S. typhimurium virulence when administered orally. Transposon mutagenesis of Proteus mirabilis also suggests that LPS modifications affect AP resistance and virulence phenotypes. Therefore, LPS modification in Gram-negative bacteria plays a significant role during infection in resistance to host antimicrobial factors, avoidance of immune system recognition, and maintenance of virulence phenotypes.     

Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases

Terminal RNA uridylyltransferases (TUTases) catalyze template-independent UMP addition to the 3' hydroxyl of RNA. TUTases belong to the DNA polymerase beta superfamily of nucleotidyltransferases that share a conserved catalytic domain bearing three metal-binding carboxylate residues. We have previously determined crystal structures of the UTP-bound and apo forms of the minimal trypanosomal TUTase, TbTUT4, which is composed solely of the N-terminal catalytic and C-terminal base-recognition domains. Here we report crystal structures of TbTUT4 with bound CTP, GTP, and ATP, demonstrating nearly perfect superposition of the triphosphate moieties with that of the UTP substrate. Consequently, at physiological nucleoside 5'-triphosphate concentrations, the protein-uracil base interactions alone are not sufficient to confer UTP selectivity. To resolve this ambiguity, we determined the crystal structure of a prereaction ternary complex composed of UTP, TbTUT4, and UMP, which mimics an RNA substrate, and the postreaction complex of TbTUT4 with UpU dinucleotide. The UMP pyrimidine ring stacks against the uracil base of the bound UTP, which on its other face also stacks with an essential tyrosine. In contrast, the different orientation of the purine bases observed in cocrystals with ATP and GTP prevents this triple stacking, precluding productive binding of the RNA. The 3' hydroxyl of the bound UMP is poised for in-line nucleophilic attack while contributing to the formation of a binding site for a second catalytic metal ion. We propose a dual role for RNA substrates in TUTase-catalyzed reactions: contribution to selective incorporation of the cognate nucleoside and shaping of the catalytic metal binding site.     

Stable three-stranded DNA made by RecA protein.

When RecA protein, in the form of a nucleoprotein filament containing circular single-stranded DNA (plus strand only), reacts with homologous linear duplex DNA, a directional transfer ensues of a strand from the duplex DNA to the nucleoprotein filament, resulting in the displacement of the linear plus strand in the 5' to 3' direction. The initial homologous synapsis, however, can occur at either end of the duplex DNA, or anywhere in between, and when homology is restricted to different regions of the duplex DNA, the joint molecules that form in each region show striking differences in stability upon deproteinization: distal joints greater than proximal joints much greater than medial joints. In the deproteinized distal joints, which are thermostable, 2000 nucleotide residues of the circular plus strand are resistant to P1 nuclease; both strands of the original duplex DNA remain resistant to P1 nuclease, and the potentially displaceable linear plus strand, which has a 3' homologous end, remains resistant to Escherichia coli exonuclease I. These observations suggest that RecA protein promotes homologous pairing and strand exchange via long three-stranded DNA intermediates and, moreover, that, once formed, such triplex structures in natural DNA are stable even when RecA protein has been removed.