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In addition to highly conserved stem-loop structures located in the 5'- and 3'-nontranslated regions, genome replication of picornaviruses requires cis-acting RNA elements located in the coding region (termed cre) (K. L. McKnight and S. M. Lemon, J. Virol. 70:1941-1952, 1996; P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999; I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). cre elements appear to be essential for minus-strand RNA synthesis by an as-yet-unknown mechanism. We have discovered that the cre element of poliovirus (mapping to the 2C coding region of poliovirus type 1; nucleotides 4444 to 4505 in 2C), which is homologous to the cre element of poliovirus type 3, is preferentially used as a template for the in vitro uridylylation of VPg catalyzed by 3D(pol) in a reaction that is greatly stimulated by 3CD(pro) (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10359-10370, 2000). Here we report a direct correlation between mutations that eliminate, or severely reduce, the in vitro VPg-uridylylation reaction and produce replication phenotypes in vivo. None of the genetic changes significantly influenced translation or polyprotein processing. A substitution mapping to the first A (A4472C) of a conserved AAACA sequence in the loop of PV-cre(2C) eliminated the ability of the cre RNA to serve as template for VPg uridylylation and abolished RNA infectivity. Mutagenesis of the second A (A4473C; AAACA) severely reduced the yield of VPgpUpU and RNA infectivity was restored only after reversion to the wild-type sequence. The effect of substitution of the third A (A4474G; AAACA) was less severe but reduced both VPg uridylylation and virus yield. Disruption of base pairing within the upper stem region of PV-cre(2C) also affected uridylylation of VPg. Virus derived from transcripts containing mutations in the stem was either viable or quasi-infectious.

The DNA binding domain of the single-stranded DNA binding protein gene V protein encoded by the bacteriophage M13 was studied by means of 1H nuclear magnetic resonance, through use of a spin-labeled deoxytrinucleotide. The paramagnetic relaxation effects observed in the 1H-NMR spectrum of M13 GVP upon binding of the spin-labeled ligand were made manifest by means of 2D difference spectroscopy. In this way, a vast data reduction was accomplished which enabled us to check and extend the analysis of the 2D spectra carried out previously as well as to probe the DNA binding domain and its surroundings. The DNA binding domain is principally situated on two beta-loops. The major loop of the two is the so-called DNA binding loop (residues 16-28) of the protein where the residues which constitute one side of the beta-ladder (in particular, residues Ser20, Tyr26, and Leu28) are closest to the DNA spin-label. The other loop is part of the so-called dyad domain of the protein (residues 68-78), and mainly its residues at the tip are affected by the spin-label (in particular, Phe73). In addition, a part of the so-called complex domain of the protein (residues 44-51) which runs contiguous to the DNA binding loop is in close vicinity to the DNA. The NMR data imply that the DNA binding domain is divided over two monomeric units of the GVP dimer in which the DNA binding loop and the tip of the dyad loop are part of opposite monomers. The view of the GVP-ssDNA binding interaction which emerges from our data differs from previous molecular modeling proposals which were based on the GVP crystal structure (Brayer & McPherson, 1984; Hutchinson et al., 1990). These models implicate the involvement of one or two tyrosines (Tyr34, Tyr41) of the complex loop of the protein to participate in complex formation with ssDNA. In the NMR studies with the spin-labeled oligonucleotides, no indication of such interactions has been found. Other differences between the models and our NMR data are related to the structural differences found when solution and crystal structures are compared.

The naturally occurring DNA-nucleopeptide H-Asp-Ser[5'-pAAAGTAAGCC-3']-Glu-OH was prepared via a solid-phase phosphite triester approach using N-2-(tert-butyldiphenylsilyloxymethyl)benzoyl protected nucleosides. The oligonucleotide was linked via the extremely base-labile oxalyl ester anchor to the solid support.

The naturally occurring RNA-nucleopeptide H-Ala-Tyr[5'-pUUAAAAC-3']-NH2 is prepared via a solid-phase phosphite triester approach using N-SiOMB/O-TBDMS-protected nucleosides. Preliminary 1H-NMR studies show that the peptidyl unit has a remarkable effect on the conformational behaviour of the RNA moiety in the nucleopeptide.

Before integration of the human immunodeficiency virus (HIV) DNA, two nucleotides are removed from the 3' ends of the viral DNA by the integrase (IN) protein. We studied the chemistry of this reaction, and found that IN mediates site-specific hydrolysis of a phosphodiester bond, resulting in release of a dinucleotide. A class of alcohols (including glycerol, 1,2-propanediol, but not 1,3-propanediol) can also act as nucleophile in this reaction, and likewise the alcoholic amino acids L-serine and L-threonine can be covalently linked to the dinucleotide. No evidence was found for a covalent linkage between the IN protein and this dinucleotide, suggesting that IN directs a single nucleophilic attack of water at the specific phosphodiester bond.

The bacteriophage phi X174 gene A protein cleaves the viral strand of the double-stranded replicative form (RF) DNA of the phage at a specific site, the origin. It leaves a free 3'-OH at nucleotide 4305 (G) of the phi X DNA sequence and binds covalently to the DNA. The nature and position of the covalent bond have been determined using the octadecadesoxyribonucleotide CAACTTG[32P]ATATTAATAAC. This octadecamer, which corresponds to nucleotides 4299-4316 of phi X viral DNA, is cleaved by gene A protein. Gene A protein is bound to the labelled phosphate via a tyrosyl residue, indicating that binding occurs to the nucleotide corresponding to 4306 (A) of the phi X viral DNA strand.

The bacteriophage phi X174 gene A encodes two proteins: gene A protein and A* protein. Purified A* protein acts as a single-stranded, DNA-specific endonuclease which remains covalently attached to the 5'-end of the cleavage site. Incubation of A* protein with the synthetic heptamer CAACTTG or with oligonucleotides which yield this heptamer after cleavage with the A* protein yields oligonucleotides with the sequences CAACTTGAG, CAACTTGAGG and CAACTTGAGGA. This indicates that A* protein carries an oligonucleotide with the sequence--AG, -AGG or -AGGA. The oligonucleotide can be transferred to the 3'-end of the heptamer CAACTTG. This suggests that A* protein reacts with a specific DNA sequence in the infected cell.

In this paper, a detailed description is presented of the aromatic part of the 500-MHZ 1H nuclear magnetic resonance (NMR) spectrum of the helix-destabilizing gene-5 protein (GVP) encoded by the coliphage M13. As a result of the resolution obtained at 500 MHZ, it was possible to perform selective decoupling and time-resolved selective Overhauser experiments. The magnitudes of the observed Overhauser effects compare favorably with magnitudes expected on the basis of theoretical calculations. These experiments in conjunction with selective decoupling experiments allowed a detailed interpretation of the aromatic part of the protein spectrum. The spectrum of the aromatic part of the GVP-d(A)8 complex could be interpreted in a similar fashion. The ring protons of one phenylalanyl residue and of two tyrosyl residues show rather large shifts upon complex formation. This indicates that these residues are involved in the interaction with the DNA molecule in accordance with earlier observations. Direct evidence for the proximity of these aromatic rings and the DNA fragment in the complex was obtained by additional Overhauser experiments. It turns out that the H3',H4', and/or the H5' sugar protons of the oligonucleotide are situated near the ring protons of (most likely) two or all three of the aromatic residues of which the resonances undergo large shifts upon complex formation.

Synthetic oligodeoxyribonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the 30 b.p. origin region of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the kanamycin resistance gene of pACYC177 (AmpR, KmR). Transformants were picked up by antibiotic selection and filter-hybridization using one of the oligodeoxyribonucleotides as a probe. Approximate lengths of the inserts were determined by restriction enzyme analysis. Exact length and orientation of each insert was determined by DNA sequencing. Plasmid DNA with an insert homologous to the first 25 b.p. of the phi X174 origin is not nicked by the gene A protein. However, plasmid DNA containing the 27 b.p. fragment in either orientation is nicked by the gene A protein, as well as plasmid DNAs containing the first 28 b.p. or the complete 30 b.p. conserved origin region of the isometric phages.

Synthetic oligonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the origin of replication of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the unique SmaI or HindIII restriction sites in the kanamycin-resistance gene of pACYC177 (AmpR, KmR). Recombinant plasmids were picked up by colony hybridization. DNA sequencing showed that not only recombinant plasmids with the expected insert were formed, but also recombinant plasmids with a shorter insert. Recombinant plasmids with an insert homologous to the first 24, 25, 26, 27, 28 or all 30 b.p. of the phi X174 origin region were thus obtained. Supercoiled plasmids containing a sequence homologous to the first 27, 28 or 30 b.p. of the phi X174 origin region are nicked by the phi X174 gene A protein. However, the other supercoiled plasmids are not nicked by the phi X174 gene A protein. These results show that the first 27 b.p. of the phi X174 origin region are sufficient as well as required for the initiation step in phi X174 RF DNA replication, i.e. the cleavage by gene A protein.

The binding of gene-5 protein to oligo(deoxyadenylic acid)s varying in length from 2 to 16 nucleotides has been studied by titrating the protein with the oligonucleotides and recording the 1H NMR spectra at 360 MHz. To obtain information about the mode of binding of the protein the aromatic parts of the spectra have been analysed by performing spectral simulations, starting from the assignments obtained from nuclear Overhausfer enhancements at 500 MHz [Alma, N. C. M., Harmsen, B. J. M., Hull, W. E., Van der Marel, G., Van Boom, J.H., and Hilbers, C. W. (1981) Biochemistry, 20, 4419-4428]. The 1H NMR spectra of the complexes of gene-5 protein with (dA)8, (dA)12 and (dA)16 appear to be identical except for differences in linewidth. The 1H NMR spectra of the complexes with the smaller oligonucleotides (dA)2, (dA)3 and (dA)4 differ from each other and from the spectra obtained from the complexes with longer oligonucleotides. However, binding of all oligonucleotides basically influences the same aromatic residues, namely two tyrosines and one phenylalanine. In the protein-oligonucleotide complexes, one protein monomer covers three nucleotide residues, in contrast to the stoichiometry of 1:4 found for protein-polynucleotide complexes. It was found that the binding to oligonucleotides is cooperative and ionic-strength-dependent but far less so than found for the binding to polynucleotides.

The interaction of gene V protein from bacteriophage M13 with the self-complementary tetranucleotide d(pC-G-C-G) was studied by 1H and 31P nuclear magnetic resonance. It is shown, using the hydrogen-bonded proton resonances of the Watson-Crick base pairs as a probe, that the protein is able to unwind the small double-helical fragment even at 0 degrees C. Binding of the tetranucleotide causes changes in the aromatic part of the 1H NMR spectrum of the complex, suggesting that aromatic residues, most likely tyrosines, take part in the protein.nucleic-acid interaction. From the 31P NMR spectra of the protein.nucleic-acid complex it follows that the pK value of the 5'-terminal phosphate is lower than for the free nucleic acid species. Moreover, it could be shown that the exchange of the protein between nucleic acid substrates is fast. Combination of these measurements has led us to derive a mechanism of unwinding on the tetranucleotide level. To a large extent the unwinding is determined by fluctuations in the double-helical DNA structure.

Gene A protein, the initiator protein of bacteriophage phi X174 DNA replication, cleaves synthetic single-stranded oligodeoxyribonucleotides at the same site as the corresponding sequence at the phi X origin. The results identify the recognition sequence within the decamer CAACTTGATA which is cleaved next to the G residue. Further requirements for cleavage of double-stranded DNA by the gene A protein are supercoiling and an A + T-rich domain adjacent to the recognition sequence.

Bacteriophage phi X174 gene A encodes two proteins: the gene A protein and the smaller A protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A protein in the life cycle of phi X174, however, is unknown. Using oligonucleotide-directed mutagenesis a viable phi X174 mutant was constructed in which the ATG start codon of the A protein was changed into an ATT codon. This mutant, phi X-4499T, does not synthesize A protein. The burst size of phi X-4499T amounted to 50% of that of wild type phi X174. This indicates that A protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage phi X174.

The complex of the gene-5 protein of bacteriophage M13 with octadeoxyadenylic acid [d(A)8] has been shown earlier to differ in various respects from the complex with polynucleotides [Alma, N. C. M., Harmsen, B. J. M., van Boom, J. H., van der Marel, G., & Hilbers, C. W. (1982) Eur. J. Biochem. 122, 319-326]. In this paper the gene-5 protein-d(A)8 complex is compared with the complex formation between the gene-5 protein and a mixture of longer oligonucleotides, i.e., d(A)25-30. Nuclear Overhauser experiments have been performed on both systems to obtain structural information regarding the oligonucleotide protein interactions. In the experiments also oligonucleotides deuterated at the adenyl C8 positions have been used in order to distinguish between the adenyl H2 and H8 resonances. Combination of the experiments shows that the nucleotides in the complexes are situated in such a way that the adenyl H8 and the sugar H1' protons are near the protein surface while the adenyl H2 protons are relatively far removed from all other protons, indicating that this side of the base is pointing away from the protein surface. It is concluded that structurally different complexes can be obtained for the d(A)25-30 system. The complex with d(A)25-30 undergoes a structural transition when going from excess oligonucleotide to excess gene-5 protein. This transition is identified with the transition between the "oligonucleotide" and the "polynucleotide" binding mode. The information derived from the present NMR experiments combined with known data from X-ray diffraction and electron microscopic studies is used to propose a model for a possible orientation of the adenyl bases in the complex.