Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs

Unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs) are relatively common in bacterial DNA but are rare in vertebrate DNA. B cells and monocytes have the ability to detect such CpG-S motifs that trigger innate immune defenses with production of Th1-like cytokines. Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus is immune-stimulatory, but serotype 2 is nonstimulatory and can even inhibit activation by bacterial DNA. In type 12 genomes, the distribution of CpG-flanking bases is similar to that predicted by chance. However, in type 2 adenoviral DNA the immune stimulatory CpG-S motifs are outnumbered by a 15- to 30-fold excess of CpG dinucleotides in clusters of direct repeats or with a C on the 5′ side or a G on the 3′ side. Synthetic oligodeoxynucleotides containing these putative neutralizing (CpG-N) motifs block immune activation by CpG-S motifs in vitro and in vivo. Eliminating 52 of the 134 CpG-N motifs present in a DNA vaccine markedly enhanced its Th1-like function in vivo, which was increased further by the addition of CpG-S motifs. Thus, depending on the CpG motif, prokaryotic DNA can be either immune-stimulatory or neutralizing. These results have important implications for understanding microbial pathogenesis and molecular evolution and for the clinical development of DNA vaccines and gene therapy vectors.

A thymidine triphosphate shape analog lacking Watson–Crick pairing ability is replicated with high sequence selectivity

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5′-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo− mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F–A pair is associated with a brief pause, whereas that beyond A in the inverted A–F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A–F/F–A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.

Sequence-specific recognition of a subgenomic RNA promoter by a viral RNA polymerase

RNA templates of 33 nucleotides containing the brome mosaic virus (BMV) core subgenomic promoter were used to determine the promoter elements recognized by the BMV RNA-dependent RNA polymerase (RdRp) to initiate RNA synthesis. Nucleotides at positions −17, −14, −13, and −11 relative to the subgenomic initiation site must be maintained for interaction with the RdRp. Changes to every other nucleotide at these four positions allow predictions for the base-specific functional groups required for RdRp recognition. RdRp contact of the nucleotide at position −17 was suggested with a template competition assay. Comparison of the BMV subgenomic promoter to those from other plant and animal alphaviruses shows a remarkable degree of conservation of the nucleotides required for BMV subgenomic RNA synthesis. We show that the RdRp of the plant-infecting BMV is capable of accurately, albeit inefficiently, initiating RNA synthesis from the subgenomic promoter of the animal-infecting Semliki Forest virus. The sequence-specific recognition of RNA by the BMV RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.

A curved RNA helix incorporating an internal loop with G·A and A·A non-Watson–Crick base pairing

The crystal structure of the RNA dodecamer 5′-GGCC(GAAA)GGCC-3′ has been determined from x-ray diffraction data to 2.3-Å resolution. In the crystal, these oligomers form double helices around twofold symmetry axes. Four consecutive non-Watson–Crick base pairs make up an internal loop in the middle of the duplex, including sheared G·A pairs and novel asymmetric A·A pairs. This internal loop sequence produces a significant curvature and narrowing of the double helix. The helix is curved by 34° from end to end and the diameter is narrowed by 24% in the internal loop. A Mn2+ ion is bound directly to the N7 of the first guanine in the Watson–Crick region following the internal loop and the phosphate of the preceding residue. This Mn2+ location corresponds to a metal binding site observed in the hammerhead catalytic RNA.

Multiple-complete-digest restriction fragment mapping: Generating sequence-ready maps for large-scale DNA sequencing

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of ×15–30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a “shotgun” method.

Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT “DNA-unwinding motif,” which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA.

A general procedure for locating and analyzing protein-binding sequence motifs in nucleic acids

In the last decade, two tools, one drawn from information theory and the other from artificial neural networks, have proven particularly useful in many different areas of sequence analysis. The work presented herein indicates that these two approaches can be joined in a general fashion to produce a very powerful search engine that is capable of locating members of a given nucleic acid sequence family in either local or global sequence searches. This program can, in turn, be queried for its definition of the motif under investigation, ranking each base in context for its contribution to membership in the motif family. In principle, the method used can be applied to any binding motif, including both DNA and RNA sequence families, given sufficient family size.

Regulation of ribonuclease III processing by double-helical sequence antideterminants

The double helix is a ubiquitous feature of RNA molecules and provides a target for nucleases involved in RNA maturation and decay. Escherichia coli ribonuclease III participates in maturation and decay pathways by site-specifically cleaving double-helical structures in cellular and viral RNAs. The site of cleavage can determine RNA functional activity and half-life and is specified in part by local tertiary structure elements such as internal loops. The involvement of base pair sequence in determining cleavage sites is unclear, because RNase III can efficiently degrade polymeric double-stranded RNAs of low sequence complexity. An alignment of RNase III substrates revealed an exclusion of specific Watson–Crick bp sequences at defined positions relative to the cleavage site. Inclusion of these “disfavored” sequences in a model substrate strongly inhibited cleavage in vitro by interfering with RNase III binding. Substrate cleavage also was inhibited by a 3-bp sequence from the selenocysteine-accepting tRNASec, which acts as an antideterminant of EF-Tu binding to tRNASec. The inhibitory bp sequences, together with local tertiary structure, can confer site specificity to cleavage of cellular and viral substrates without constraining the degradative action of RNase III on polymeric double-stranded RNA. Base pair antideterminants also may protect double-helical elements in other RNA molecules with essential functions.

Characterization of BseMII, a new type IV restriction–modification system, which recognizes the pentanucleotide sequence 5′-CTCAG(N)10/8↓

We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5′-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3′-protruding end. Magnesium ions and S-adenosyl-l-methionine (AdoMet) are required for cleavage. S-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5′-CTCAG-3′/5′-CTGAG-3′. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5′-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-l-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.

Kinetics of formation of hypoxanthine containing base pairs by HIV-RT: RNA template effects on the base substitution frequencies

Hypoxanthine (H), the deamination product of adenine, has been implicated in the high frequency of A to G transitions observed in retroviral and other RNA genomes. Although H·C base pairs are thermodynamically more stable than other H·N pairs, polymerase selection may be determined in part by kinetic factors. Therefore, the hypoxanthine induced substitution pattern resulting from replication by viral polymerases may be more complex than that predicted from thermodynamics. We have examined the steady-state kinetics of formation of base pairs opposite template H in RNA by HIV-RT, and for the incorporation of dITP during first- and second-strand synthesis. Hypoxanthine in an RNA template enhances the k2app for pairing with standard dNTPs by factors of 10–1000 relative to adenine at the same sequence position. The order of base pairing preferences for H in RNA was observed to be H·C >> H·T > H·A > H·G. Steady-state kinetics of insertion for all possible mispairs formed with dITP were examined on RNA and DNA templates of identical sequence. Insertion of dITP opposite all bases occurs 2–20 times more frequently on RNA templates. This bias for higher insertion frequencies on RNA relative to DNA templates is also observed for formation of mispairs at template A. This kinetic advantage afforded by RNA templates for mismatches and pairing involving H suggests a higher induction of mutations at adenines during first-strand synthesis by HIV-RT.

Complete genomic sequence of Pasteurella multocida,Pm70

We present here the complete genome sequence of a common avian clone of Pasteurella multocida, Pm70. The genome of Pm70 is a single circular chromosome 2,257,487 base pairs in length and contains 2,014 predicted coding regions, 6 ribosomal RNA operons, and 57 tRNAs. Genome-scale evolutionary analyses based on pairwise comparisons of 1,197 orthologous sequences between P. multocida, Haemophilus influenzae, and Escherichia coli suggest that P. multocida and H. influenzae diverged ≈270 million years ago and the γ subdivision of the proteobacteria radiated about 680 million years ago. Two previously undescribed open reading frames, accounting for ≈1% of the genome, encode large proteins with homology to the virulence-associated filamentous hemagglutinin of Bordetella pertussis. Consistent with the critical role of iron in the survival of many microbial pathogens, in silico and whole-genome microarray analyses identified more than 50 Pm70 genes with a potential role in iron acquisition and metabolism. Overall, the complete genomic sequence and preliminary functional analyses provide a foundation for future research into the mechanisms of pathogenesis and host specificity of this important multispecies pathogen.

Complete genome sequence of Caulobacter crescentus

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living α-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

SfiI endonuclease activity is strongly influenced by the non-specific sequence in the middle of its recognition site

The SfiI endonuclease cleaves DNA at the sequence GGCCNNNN↓NGGCC, where N is any base and ↓ is the point of cleavage. Proteins that recognise discontinuous sequences in DNA can be affected by the unspecified sequence between the specified base pairs of the target site. To examine whether this applies to SfiI, a series of DNA duplexes were made with identical sequences apart from discrete variations in the 5 bp spacer. The rates at which SfiI cleaved each duplex were measured under steady-state conditions: the steady-state rates were determined by the DNA cleavage step in the reaction pathway. SfiI cleaved some of these substrates at faster rates than other substrates. For example, the change in spacer sequence from AACAA to AAACA caused a 70-fold increase in reaction rate. In general, the extrapolated values for kcat and Km were both higher on substrates with inflexible spacers than those with flexible structures. The dinucleotide at the site of cleavage was largely immaterial. SfiI activity is thus highly dependent on conformational variations in the spacer DNA.

Enhancement of translation by the epsilon element is independent of the sequence of the 460 region of 16S rRNA

The epsilon enhancer element is a pyrimidine-rich sequence that increases expression of T7 gene 10 and a number of Escherichia coli mRNAs during initiation of translation and inhibits expression of the recF mRNA during elongation. Based on its complementarity to the 460 region of 16S rRNA, it has been proposed that epsilon exerts its enhancer activity by base pairing to this complementary rRNA sequence. We have tested this model of enhancer action by constructing mutations in the 460 region of 16S rRNA and examining expression of epsilon-containing CAT reporter genes and recF–lacZ fusions in strains expressing the mutant rRNAs. Replacement of the 460 E.coli stem–loop with that of Salmonella enterica serovar Typhimurium or a stem–loop containing a reversal of all 8 bp in the helical region produced fully functional rRNAs with no apparent effect on cell growth or expression of any epsilon-containing mRNA. Our experiments confirm the reported effects of the epsilon elements on gene expression but show that these effects are independent of the sequence of the 460 region of 16S rRNA, indicating that epsilon–rRNA base pairing does not occur.

Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication

The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III–box I hairpin with a 3′ single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III–box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III–box I hairpin in partially unwound DNA.