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Unlike most viral assembly systems, two scaffolding proteins, B and D, mediate bacteriophage øX174 morphogenesis. The external scaffolding protein D is highly ordered in the atomic structure and proper function is very sensitive to mutation. In contrast, the internal scaffolding protein B is relatively unordered and extensive alterations do not eliminate function. Despite this genetic laxity, protein B is absolutely required for virus assembly. Thus, this system, with its complex arrangements of overlapping reading frames, can be regarded as an example of "irreducible complexity." To address the biochemical functions of a dual scaffolding protein system and the evolution of complexity, progressive and targeted genetic selections were employed to lessen and finally eliminate B protein-dependence. The biochemical and genetic bases of adaptation were characterized throughout the analysis that led to the sextuple mutant with a B-independent phenotype, as evaluated by plaque formation in wild-type cells. The primary adaptation appears to be the over-expression of a mutant external scaffolding protein. Progeny production was followed in lysis-resistant cells. The ability to produce infectious virions does not require all six mutations. However, the lag phase before progeny production is shortened as mutations accumulate. The results suggest that the primary function of the internal scaffolding protein may be to lower the critical concentration of the external scaffolding protein needed to nucleate procapsid formation. Moreover, they demonstrate a novel mechanism by which a stringently required gene product can be bypassed, even in a system encoding only eight strictly essential proteins.

On linear single-stranded DNA, RecA filaments assemble and disassemble in the 5' to 3' direction. Monomers (or other units) associate at one end and dissociate from the other. ATP hydrolysis occurs throughout the filament. Dissociation can result when ATP is hydrolyzed by the monomer at the disassembly end. We have developed a comprehensive model for the end-dependent filament disassembly process. The model accounts not only for disassembly, but also for the limited reassembly that occurs as DNA is vacated by disassembling filaments. The overall process can be monitored quantitatively by following the resulting decline in DNA-dependent ATP hydrolysis. The rate of disassembly is highly pH dependent, being negligible at pH 6 and reaching a maximum at pH values above 7. 5. The rate of disassembly is not significantly affected by the concentration of free RecA protein within the experimental uncertainty. For filaments on single-stranded DNA, the monomer kcat for ATP hydrolysis is 30 min-1, and disassembly proceeds at a maximum rate of 60-70 monomers per minute per filament end. The latter rate is that predicted if the ATP hydrolytic cycles of adjacent monomers are not coupled in any way.

The assembly of the viral structural proteins into infectious virions is often mediated by scaffolding proteins. These proteins are transiently associated with morphogenetic intermediates but not found in the mature particle. The genes encoding three Microviridae (phiX174, G4 and alpha3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species. Despite divergence as great as 70% in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles. These results suggest that the Microviridae internal scaffolding proteins may be inherently flexible. There was one condition in which a B protein could not cross-function. The phiX174 B protein cannot productively direct the assembly of the G4 capsid at temperatures above 21 degreesC. Under these conditions, assembly is arrested early in the morphogenetic pathway, before the first B protein mediated reaction. Two G4 mutants, which can productively utilize the phiX174 B protein at elevated temperatures, were isolated. Both mutations confer amino acid substitutions in the viral coat protein but differ in their relative abilities to utilize the foreign scaffolding protein. The more efficient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region.

Bacteriophage oX174 morphogenesis requires two scaffolding proteins: an internal species, similar to those employed in other viral systems, and an external species, which is more typically associated with satellite viruses. The current model of oX174 assembly is based on structural and in vivo data. During morphogenesis, 240 copies of the external scaffolding protein mediate the association of 12 pentameric particles into procapsids. The hypothesized pentameric intermediate, the 12S⁎ particle, contains 16 proteins: 5 copies each of the coat, spike and internal scaffolding proteins and 1 copy of the DNA pilot protein. Assembly naive 12S⁎ particles and external scaffolding oligomers, most likely tetramers, formed procapsid-like particles in vitro, suggesting that the 12S⁎ particle is a bona fide assembly intermediate and validating the current model of procapsid morphogenesis. The in vitro system required a crowding agent, was influenced by the ratio of the reactants and was most likely driven by hydrophobic forces. While the system reported here shared some characteristics with other in vitro internal scaffolding protein-mediated systems, it displayed unique features. These features most likely reflect external scaffolding protein-mediated morphogenesis and the oX174 procapsid structure, in which external scaffolding-scaffolding protein interactions, as opposed to coat-coat protein interactions between pentamers, constitute the primary lattice-forming contacts.

Like most bacteriophages, phi X174 transfers its DNA through the cell wall, leaving an empty capsid on the cell surface. The process begins with ejection of the genome at its host-receptor site. The rate of this event can be measured, so detailed structure/function analysis of the mechanism is possible now that an atomic structure of the phi X174 protein shell has been obtained. Amino acid substitutions at two arginine residues near the DNA-binding pocket of F capsid protein decrease the eclipse rate, while deletion of 27 bases from the J-F non-coding region increases the rate. An alanine to serine change in the N-terminal region of the phi X174 H "spike" protein has suppressor activity in that this mutation also increases the eclipse rate when the complete genome is present within both mutant and wild-type F capsids. These results suggest that a portion of H protein is inside the capsid, and disruption of DNA-protein interactions is involved in the ejection mechanism.

A selective adsorption method is presented which allows a rapid separation of phiX174 replicative form DNA from single-stranded phiX174 phage DNA, facilitating the handling of a large number of samples. The single-stranded phiX174 DNA is selectively adsorbed to silicic acid coated with methylated albumin. After removal of the adsorbent by sedimentation, at least 40% of the input replicative form DNA is recovered in the supernatant fraction; the ratio of replicative form to single-stranded phage DNA is increased 20- to 200-fold. The potential of the method for separating other nucleic acids is discussed.

As already known, phiX174 replicative-form DNA is separated into two components when sedimented in a sucrose gradient. The faster component, I, is not denatured by heat treatment, whereas the slower component, II, is converted to single strands. Only the infectivity of component II increases after heat denaturation. A quantitative comparison of data obtained by band centrifugation and by electron microscopy shows that the tertiary structure of component I is a highly twisted circle and that of component II an extended circle. By treatment with DNase, component I (as observed in band centrifugation) and twisted rings (as observed by electron microscopy) are converted at the same rate to component II and to extended circles and linear molecules. The plating efficiency of replicative-form DNA, in contrast to denatured component II replicative form and to phiX174 single-stranded DNA, is diminished by the addition of Escherichia coli DNA to the adsorption medium. The inhibition by E. coli DNA at concentrations above 0.5 microg/ml. is of first order. The lower the relative plating efficiency of native replicative-form DNA, the greater is the increase of infectivity for the denatured component II. The importance of these findings with respect to normal test conditions is discussed. Some observations suggest that under ideal test conditions the relative plating efficiency of the replicative-form DNA may rise to that of phiX174 single-stranded DNA.

fX174 utilizes two scaffolding proteins during morphogenesis, an internal protein (B) and an external protein (D). The B protein induces a conformational change in coat protein pentamers, enabling them to interact with both spike and external scaffolding proteins. While functions of the carboxyl terminus of protein B have been defined, the functions of the amino terminus remain obscure. To investigate the morphogenetic functions of the amino terminus, several 5 0 deleted genes were constructed and the proteins expressed in vivo. The DNH2 B proteins were assayed for the ability to complement an ochre B mutant and defects in the morphogenetic pathway were characterized. The results of the biochemical, genetic and second-site genetic analyses indicate that the amino terminus induces conformational changes in the viral coat protein and facilitates minor spike protein incorporation. Defects in conformational switching can be suppressed by substitutions in the external scaffolding protein, suggesting some redundancy of function between the two proteins. q 2003 Elsevier Ltd. All rights reserved.

An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage phiX174. Assembly of the phiX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a "closed" procapsid particle has been determined to 3.5 A resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A resolution electron microscopic reconstruction of the "open" procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly alpha-helical fold and displays remarkable conformational variability. We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.

Bacteriophage G4 and phiX174 are members of the Microviridae family. The degree of similarity of the structural proteins ranges from 66% identity of the F protein to 40% identity of the G protein. The atomic structure of the phiX174 virion had previously been determined by X-ray crystallography. Bacteriophage G4 procapsids, consisting of the structural proteins F, G, D, B, H, and small traces of J but no DNA, were set up for crystallization. However, the resultant crystals were of degraded procapsid particles, which had lost the assembly scaffolding proteins D and B, resulting in particles that resembled empty virions. The structure of the degraded G4 procapsid has been determined to 3.0 angstrom resolution. The particles crystallized in the hexagonal space group P6(3)22 with unit cell dimensions a=b=414.2(5) angstrom and c=263.0(3) angstrom. The diffraction data were collected at the Cornell High Energy Synchrotron Source (CHESS) on film and image plates using oscillation photography. Packing considerations indicated there were two particles per unit cell. A self-rotation function confirmed that the particles were positioned on 32 point group special positions in the unit cell. Initial phases were calculated to 6 angstrom resolution, based on the known phiX174 virion model. Phase information was then extended in steps to 3.0 angstrom resolution by molecular replacement electron density modification and particle envelope generation. The resulting electron density map was readily interpretable in terms of the F and G polypeptides, as occur in the mature capsid of phiX174. In a few regions of the electron density map there were inconsistencies between the density and the published amino acid sequence. Redetermining the amino acid sequence confirmed that the density was correct. The r.m.s. deviation between the Calpha backbone of the mature capsid of phiX174 and the degraded G4 procapsid was 0.36 angstrom for the F protein and 1.38 angstrom for the G protein. This is consistent with the greater conservation of the F protein compared to the G protein sequences among members of the Microviridae family. Functionally important features between phiX174 and G4 had greater conservation. Calcium ions (Ca2+) were shown to bind to G4 at a general site located near the icosahedral 3-fold axis on the F protein capsid, equivalent to sites found previously in phiX174. Binding of Ca2+ also caused the ordering of the conserved region of the DNA binding protein J, which was present in the degraded procapsid particle in the absence of DNA.

Monoclinic P2(1) crystals of the bacteriophage phi X174 have been incubated with calcium ions (Ca2+) and the induced structural conformational changes studied to 3 A resolution with X-ray crystallographic methods. Three different types of Ca2+ binding sites have been located within the asymmetric unit of the virion. Two sets of sites are associated with the F capsid protein. One set of sites associated with the F protein is in a general position near the icosahedral 3-fold axes of the virus, with the main-chain carbonyl oxygen atoms of residues Gly1321, Asp1421, Met1424 and Ser1426, and the side-chains of Gln1004 and Asp1421 as ligands. The other set of sites associated with the F protein is on the icosahedral 3-fold axes, with the symmetry-related main-chain carbonyl oxygen atoms of Ser1001 and the side-chains of Asn1002 as ligands. The bound Ca2+ induce a conformational change of the amino-terminal residues of the F proteins. A third set of sites, consisting of a pair of Ca2+ on the icosahedral 5-fold axes, are associated with the G spike protein and are concurrently liganded by the symmetry-related carbonyl oxygen side-chains of Asp2117. Concomitant with the binding of Ca2+ to the phage is the rotation of the Asp1209 side-chain of the F protein towards some additional electron density that was not observed in the absence of Ca2+. This density is situated in a shallow depression near the icosahedral 2-fold axes of the virus, and has been tentatively interpreted as a bound glucose molecule that is ordered only in the presence of Ca2+. The putative glucose binding site may be related to the attachment of the virus to cell surface lipopolysaccharides in the initial stages of Escherichia coli infection.

The structure of the bacteriophage phi X174 was examined in a 2.7 A resolution map and refined, using 6.0 A to 3.0 A resolution data with For = 5 sigma (F). The final R-factor was 20.9% and the root-mean-square deviation from idealized bond lengths was 0.021 A. The Hendrickson-Konnert refinement was restrained by the phases derived from the molecular replacement icosahedral averaging procedure. The mature phage capsid consists of 60 copies of the F protein with 426 amino acids, the G protein with 175 amino acids and the J protein with 37 amino acids, as well as 12 copies of the H protein with 328 amino acids. The entire polypeptide chain of the F and G protein, all but the first N-terminal residue of the J protein, and 178 solvent molecules were included in the refinement calculations. The secondary structural features of the F, G and J proteins and their interactions with each other are described. The majority of the protein-protein interactions are between the icosahedral 5-fold related interfaces of the F and of the G proteins. These pentameric units of the F and G proteins form the 9S and 6S assembly intermediates, respectively. The J protein lacks any secondary structure and acts as a linking arm between the icosahedral 5-fold related F proteins. Water molecules were introduced only after phase extension to 2.7 A resolution had been completed. The F protein is associated with lower "thermal" parameters and exhibits greater water order in its environment than the G and J proteins. The largest thermal parameters occur in residues on the viral surface. The solvent contributes to the interactions between the proteins. There is an interface of solvent molecules between the F and the G pentamers which stabilizes the pentameric G protein spikes in a crater centered at each of the icosahedral 5-fold vertices of the F protein capsid. Sequence alignments of the F, G and J amino acid sequences for the homologous bacteriophages G4, alpha 3, phi K and phi X174 with respect to the phi X174 structure demonstrated the conservation of functionally important residues on the viral surface.

We demonstrate that the step of DNA strand exchange during RecA-mediated recombination reaction can occur equally efficiently in the presence or absence of ATP hydrolysis. The polarity of strand exchange is the same when instead of ATP its non-hydrolyzable analog adenosine-5'-O-(3-thiotriphosphate) is used. We show that the ATP dependence of recombination reaction is limited to the post-exchange stages of the reactions. The low DNA affinity state of RecA protomers, induced after ATP hydrolysis, is necessary for the dissociation of RecA-DNA complexes at the end of the reaction. This dissociation of RecA from DNA is necessary for the release of recombinant DNA molecules from the complexes formed with RecA and for the recycling of RecA protomers for another round of the recombination reaction.

Crystals of the single-stranded DNA bacteriophage φX174 have been grown. They have a monoclinic unit cell with space group P 2 1 , unit cell dimensions of a = 306.0 (±0.2) A , b = 361.1 (±0.2) A , c = 299.7 (±0.2°) a A , β = 92.91° (±0.02°) and diffract to at least 2.7 A resolution. There are two virus particles per unit cell. Packing considerations show that the mean diameter of the virus particles is 280 A. The virus separates into two bands in a sucrose gradient. The ratio between the absorbance at 260 nm and 280 nm is 1.45 to 1.65 for the faster and 1.15 to 1.35 for the slower bands, but both bands contain intact particles. Crystals derived from these bands are isomorphous and there is no detectable difference in their structure amplitudes.

The single-stranded DNA binding protein of Ustilago maydis decreases the contour length of φX174 DNA. When DNA complexes were prepared with subsaturating amounts of the protein, its distribution on the DNA was markedly non-random, indicating a high degree of co-operativity in its binding to single-stranded DNA. The analagous Escherichia coli, Salmonella typhimurium and bacteriophage T7 binding proteins also reduced DNA contour lengths to a similar extent, whereas the bacteriophage T4 gene 32 protein, as shown previously, increased the contour length. Despite the fact that the U. maydis protein efficiently denatures poly[d(A-T) · d(A-T)], it appears to initiate denaturation of native bacteriophage λ DNA rather inefficiently.

Bacteriophage G4 has the same A B and D E overlapping gene systems as φX174 and together with the A and C K overlapping gene system (Shaw et al., 1978), 7 of the 11 G4 and φX174 genes are involved in overlaps. The nucleotide differences between G4 and φX174 in the overlapping portions of the A, C and D genes are 23%, 27% and 21%, respectively, compared with 32%, 36% and 34% in the non-overlapping portions of the same genes. The amino acid differences between the G4 and φX174 overlapping B, K and E proteins, are 44%, 39% and 44%, respectively, compared with 28%, 26% and 16% in the regions of genes A, A and C, and D which contain genes B, K and E. These results suggest that the nucleotide sequences of overlapping genes evolve at almost the same rate as in non-overlapping genes, and that this is made possible by a lower amino acid sequence stringency of one of the pairs of proteins. The overlapping D E and A and C K gene systems may have originated by taking advantage of a high incidence of T nucleotides in the second codon position to produce a hydrophobic protein and the A B gene system may have evolved by read-through of the A gene into the B gene. From the nucleotide sequences, other overlapping genes appear to be possible in these bacteriophages.

The extracellular form of bacteriophage phi X174 consists of single-stranded DNA within an icosahedral capsid, which has short spikes at each of its vertices. Each spike is composed of gene G and H proteins, while the capsid itself consists of gene F protein. Since several molecules of gene H protein are injected into the cell along with the DNA, specific protein--protein and DNA--protein interactions must be broken when the genome exits and leaves an intact capsid structure at the receptor site. To demonstrate this we examined the eclipse (DNA ejection) reaction with two types of phi X174 mutants. The first contains missense mutations in a capsid or spike protein gene, and the second involves insertions or deletions in non-coding regions of the DNA. Using an improved procedure, the eclipse rate in vivo of the eclipse mutants Fcs70 has been redetermined over a larger temperature range than in previous studies. The three- to fivefold decrease in rate between 37 degrees C and 25 degrees C is due to an increase in both the enthalpy and entropy of activation when compared to the wild-type values of these kinetic parameters. This missence mutation also confers an increase in virus stability in 2 to 3 M-urea. In contrast to this, inserting 163 bases into the length of DNA packaged within the phi X174 capsid does not lead to a detectable change in eclipse rate over the same temperature range. yet this insertion into the J--F intercistronic region imparts a significant decrease in virus stability in urea. These results suggest that a specific set of non-covalent interactions is involved in phi X174 DNA ejection. This is supported by the small (50%), but significant, increase in eclipse rate that occurs when 27 bases are deleted from the J--F intercistronic region. The latter effect must be base-sequence-specific since no change in rate is observed when only seven of the 27 bases are deleted. Thus, the kinetics of the phi X174 eclipse reaction can be used as a sensitive probe of quaternary structure by correlating the change in reaction rate with alterations in amino acid and base sequences in the structural components of the virus.

Seven different revertants of bacteriophage phi X174am16 (AB5276G leads to T) have been isolated and the nature of the reversions determined by sequencing their DNA. The revertants each differ from am16 by just a single base substitution. These may be distinguished with varying degrees of ease by characteristic temperature sensitivities of growth. This has facilitated the determination of the frequency at which DNA polymerase III catalyses different types of substitution mutations in copying phi X174 DNA in vitro and in vivo. During the replicative form (RF) leads to single-stranded (SS) stage of replication in vitro, four different revertants may be readily produced according to well-defined rate laws on biasing the concentrations of dNTPs. Transversion mutations are found to be formed predominantly by purine x purine mismatching, whilst transitions are formed predominantly by G x T mismatching. The substitutions via G x T and G x A mismatches are estimated to occur at similar frequencies in vivo. The two most common revertants isolated in vivo, however, are not those readily produced during the RF leads to SS stage in vitro but are those produced on purine x purine mismatching in the SS leads to RF stage. The accuracy of the DNA polymerase in vitro appears to be similar to that in this stage in vivo. However, the overall accuracy of the RF leads to SS replication in vivo is more accurate than predicted from the measurements of the accuracy in vitro.

Six different synthetic deoxyhexadecamers complementary to the origin of bacteriophage φX174, corresponding to nucleotides 4299 to 4314, except for one preselected nucleotide change were used as primers for DNA synthesis on wild-type φX † DNA as a template. DNA synthesis was performed with Escherichia coli DNA polymerase I (Klenow fragment) in the presence of DNA ligase. Heteroduplex RFIV DNA was isolated and, after limited digestion with DNAase I, complementary strands containing the mutant primers were isolated. The biological activity of these complementary strands was assayed in spheroplasts. Spheroplasts were made from E. coli K58 ung − (uracil N -glycosylase) to prevent degradation of the complementary strands caused by uracil incorporation (Baas et al. , 1980 a ). Using (5′- 32 P) end-labeled primers, it was shown that all tested DNA polymerase preparations, including phage T4 DNA polymerase, contained variable amounts of 5′ → 3′ exonuclease activity. This nick translation activity may result in removal of the mutation in the primers, and therefore in isolation of wild-type complementary DNA instead of mutant complementary DNA. Restriction enzyme analysis of completed RFIV DNA showed that the primers can initiate DNA synthesis at more than one place on the φX174 genome. These complications result in a mixed population of complementary strand DNAs synthesized in vitro . Nevertheless, the desired mutants were picked up with high frequency using a selection test that is based on the difference in ultraviolet light sensitivity of homoduplex and heteroduplex φX174 RF DNA. Heteroduplex φX174 RF DNA is two to three times more sensitive to ultraviolet light irradiation than is homoduplex φX174 RF DNA (Baas & Jansz, 1971,1972). Phage DNA derived from single plaque lysates of two of the six mutant complementary strand DNA preparations yielded, after annealing with wild-type complementary strand DNA, heteroduplex DNA with high frequency. DNA sequence analysis in the origin region of RF DNA obtained from these two phage preparations revealed the presence of the expected mutation. RFI DNA of these two origin mutants was nicked by φX174 gene A protein in the same way as wild-type φX174 RFI DNA. Phage DNA derived from single plaque lysates of the other four mutant complementary strand DNA preparations yielded exclusively homoduplex DNA after annealing with wild-type complementary strand DNA. It is concluded that priming with these deoxyhexadecamers resulted in the synthesis of complementary φX174 DNA with lethal mutations. The implications of these results, the construction of two silent, viable φX174 origin mutants and the failure to detect four others, for the initiation mechanism of φX174 RF DNA replication are discussed.

The restriction endonuclease Mbo II, isolated from Moraxella bovis (ATCC 10900), cleaves bacteriophage φ X174 am 3 replicative form I DNA into ten fragments. The physical map of these fragments has been aligned with the sequence of φX174 DNA. There is no sequence with 2-fold rotational symmetry common to the region of all ten cleavage sites. However, the non-symmetrical sequence 5′-G-A-A-G-A-3′ 3′-C-T-T-C-T-5′ occurs near to each cleavage site. Precise mapping of the cleavages in both DNA strands at several sites places the cuts eight nucleotides to the right of the upper sequence and seven nucleotides to the right of the lower sequence.

Eukaryotic type 1 DNA topoisomerases break single-stranded DNA at specific sites. A preferred site for rat liver topoisomerase breakage in single-stranded φX174 DNA was located within a region of the DNA with the potential for duplex formation. To investigate the relationship between sites of breakage in duplex and single-stranded DNA, a restriction fragment containing sequences from the transcriptional regulatory and enhancer region of the simian virus 40 genome was used as a substrate for topoisomerase. While different patterns of breakage in the native and denatured forms of the DNA were observed, some sites of breakage were common to both forms. The break sites in the denatured DNA were a subset of the break sites in the duplex DNA and were located in regions which had the potential for intrastrand base-pairing due to distal complementary sequences. A series of single-stranded fragments were generated with the distal complementary sequences deleted and these fragments were used as substrates for topoisomerase breakage. The lack of detectable breakage at a site when the complementary sequence was deleted, suggests that topoisomerase acts at duplex regions in the single-stranded DNA and that it is not active on regions of single-stranded DNA that are not base-paired.

Expression of the φX174 lysis (E) gene, a member of an overlapping gene pair, appears to depend on a frameshift-induced chain termination by ribosomes translating the upstream D gene. A — 1 reading frameshift, possibly induced by misreading of an alanine codon as a doublet, causes ribosomes to terminate translation at two different sites, suggesting two modes of regulating expression of the E gene. One frameshift can cause translational termination at a stop codon(s) near the E gene ribosome binding site (RBS), resulting in reinitiation by ribosomes at the E gene RBS. Termination at a second site some 70 bases upstream from the E gene RBS, while too far away to allow ribosomal re-initiation at the E gene RBS, probably results in an unmasking of the message, allowing entry of a new ribosome at the E gene RBS.

By inserting palindromes of varying length and sequence into a non-essential region of the bacteriophage phi X174 genome we have investigated the effect of palindrome size and sequence on their genetic stability. Multimers of increasing size of the EcoRI linker CCGAATTCGG (E), the BamHI linker CCGGATCCGG (B) or mixtures of both (E, B) were inserted into the PvuII site of a previously constructed bacteriophage strain phi X174 J-F ins6. The largest inserts that could be maintained in the genome without significant loss of genetic stability were 2B, 4E, and 4(E, B), respectively. Polymers exceeding this size could be inserted but resulted in rapid and precise deletion from the phage genome, whereby nB was more unstable than nE, and nE was more unstable than n(E, B). Analysis of the resulting deletion mutants provided evidence for two different types of deletions. The more frequent deletion arose from either type palindrome and removed nucleotides in blocks of ten base-pairs (one linker unit), but only from the palindromic sequence, and always left at least an 18 base-pair long palindrome (one linker plus 8 neighboring base-pairs) behind. The less frequently occurring deletions arose only from nB type palindromes, removing the complete palindromic sequence plus adjacent nucleotides. At least the first type of deletion occurred in the absence of recA activity. Our results show a correlation between the sequence, as well as size, and the genetic stability of palindromes, i.e. sequences that could decrease the stability of a cruciform increased their genetic stability. This supports the theory that palindrome deletion occurs via extrusion of the palindrome into a cruciform or cruciform-like structure.

Standard formamide/cytochrome c spreading conditions, modified by the addition of 0·5 m -ammonium acetate, have been used to visualize reproducible structures resulting from long range base pairing in single-stranded DNA from bacteriophage φX174. The most highly folded circular viral DNA molecules appeared as three-lobed structures, and one or two of these lobes were also found in less folded molecules. The lobes defined three structural domains of characteristic size and morphology. The locations of these domains and seven of the stem and loop structures comprising them have been mapped relative to the molecular ends of denatured Pst I-cleaved replicative form DNA. Their orientation relative to the φX174 genetic map and nucleotide sequence was determined using denatured Hpa I-cleaved replicative form DNA. An examination of the nucleotide sequence in regions corresponding to the observed structures revealed potential base pairing with calculated negative free energies from −21 to −46 kcal/mol. The total length of the paired regions averaged 33 base-pairs, but the paired segments include no more than 11 consecutive base-pairs (including G.T pairs) interspersed with unpaired DNA of varying length. The observed structures and proposed base-paired sequences bring the 5′ and 3′ ends of linear φX174 DNA, cleaved at the origin of viral strand replication, close together which may facilitate their rejoining by φX174 gene A protein following DNA replication. In some but not all of the φX174 messenger RNA transcripts the ribosome binding sites for genes C and E may be involved in base pairing which might in part account for the relatively small quantities of their polypeptide products synthesized in vivo .

The mechanism of release of φX174 by infected E. coli is of interest since its understanding must result in the discovery of either: (1) a phage-specific lytic enzyme (Streisinger, Mukai, Dreyer, Miller & Horiuchi, 1961; Jacob & Fuerst, 1958) which would be the first example of a φX-specific enzyme; or (2) a novel method of phage release. Recently some unsuccessful attempts to detect a lytic enzyme in the mature phage particle and in infected cells have been reported (Fujimura & Kaesberg, 1962; Eigner, Stouthamer, Van der Sluys & Cohen, 1963). It has been suggested (Eigner et al., 1963) that release of φX precedes cellular dissolution. Denhardt (1963, in preparation) has shown that even when the infection process is synchronized by infection in the presence of 0·003 M-KCN or by 'infection of starved cells phage release occurs over a period of time longer than the minimum latent period. These results suggested the possibility that ,PX might be released slowly from single infected complexes, as is the case with certain animal viruses (see, for example, Dulbecco & Vogt, 1954), rather than in bursts occurring at the time of lysis.

Monomers of purified RecA protein polymerize into helical fibers whose pitch is 7.2 nm to 7.5 nm and whose diameter is 11 nm. Either short (approximately 0.2 micron), single fibers, or bundles of aligned, longer fibers, can be formed preferentially, by varying the Mg2+ concentration. When RecA protein is bound to circular, single-stranded phi X174 DNA it forms helical fibers of different classes of contour lengths, ranging from 0.98 micron, depending upon the conditions of assembly. Two different helical pitches are found, one of 9.3 nm when the incubation buffer contains, besides the obligatory Mg2+, either ATP gamma S or ATP accompanied by single-strand binding protein, and one of 5.5 nm when the latter additives are omitted. Preformed fibers of the compact type can be converted to open ones of 9.3 nm pitch upon addition of ATP gamma S, even after the removal of unbound RecA. All signs of helicity are obliterated upon glutaraldehyde cross-linking except in those fibers whose assembly has been mediated by ATP gamma S. RecA protein and single-strand binding protein are competitively bound to single-stranded DNA. Composite complexes, however, are not encountered unless ATP gamma S is present. Otherwise, segments of DNA that are coated by one or the other protein are seen as separate regions. When the assembly of complexes of single-stranded DNA and RecA is mediated by single-strand binding protein and ATP, the axial separation between successive bases is 0 X 42 nm, somewhat greater than the axial distance between bases in one strand of duplex DNA in the B form. It is proposed that the bases of the single-stranded DNA in the complex are located near its inner surface, and that base-pairing with double-stranded DNA takes place following invasion of the central cavity of the complex.

The nature of specific DNA sequences that arrest synthesis by mammalian DNA polymerase alpha in vitro was analyzed using circular, single-stranded M13 or phi X174 virion DNA templates annealed to a unique, terminally labeled, DNA primer. This method rigorously defined both the starting nucleotide position and the direction of synthesis, as well as making the amount of radioactivity proportional to the number rather than the length of nascent DNA chains. The precise nucleotide locations of arrest sites were determined over templates with complementary sequences by cloning unique DNA restriction fragments into M13 DNA and isolating virions containing either the Watson or Crick strand. Results were correlated with the locations of palindromic (self-complementary) sequences, repeated sequences, and repeated sequences with mirror-image orientation. Two classes of DNA synthesis arrest sites were identified, distinct in structure but equivalent in activity. Class I sites consisted of palindromic sequences that formed a stable hairpin structure in solution and arrested DNA polymerase on both complementary templates. The polymerase stopped precisely at the base of the duplex DNA stem, regardless of the direction from which the enzyme approached. Class II sites consisted of non-palindromic sequences that could not be explained by either secondary structure or sequence symmetry elements, and whose complementary sequence was not an arrest site. Size limits, orientation and some sequence specificity for arrest sites were suggested by the data. Arrest sites were also observed in vivo by mapping the locations of 3'-end-labeled nascent simian virus 40 DNA strands throughout the genome. Arrest sites closest to the region where termination of replication occurs were most pronounced, and the locations of 80% of the most prominent sites appeared to be recognized by alpha-polymerase on the same template in vitro. However, class I sites were not identified in vivo, suggesting that palindromic sequences do not form hairpin structures at replication forks.

The fidelity of DNA replication in vitro by DNA polymerase I (large subfragment) of Escherichia coli has been measured by the standard bioassay: single-stranded φX174 DNA ( plus strand) containing an amber codon was primed with a synthetic oligodeoxynucleotide, replicated and the frequency of point mutations formed in the synthetic minus strand of the resultant double-stranded DNA determined from the number of revertant phage produced in a spheroplast assay. Since the assay depends crucially on the frequency of expression of the mutations in the heteroduplex, and this can vary for a variety of reasons, parallel control experiments were performed using a primer that covered the amber codon but contained the same mismatch that occurred during replication. The frequency of expression of these mutations was found to vary from 40 to 100% in fully ligated heteroduplexes, depending upon the age and batch of spheroplasts used. The variation probably reflects the viability of the post-replicative mismatch repair enzymes in the spheroplasts used for transfection. Far lower frequencies of expression were found under conditions of poor replication. Accurate data and rate laws for fidelity are obtained only when the bioassay is normalized for the variation in the expression frequency. There is active proofreading by the 3′-5′-exonuclease activity of the polymerase of a misincorporation resulting from a dGTP: T mismatch. The contribution of proofreading to fidelity is low: accuracy is enhanced by a factor of less than 7 at the concentrations of dNTPs in vivo . The lower accuracy of Pol I than Pol III is due mainly to poorer proofreading, which is manifested in a lower “cost” of replication: only 0.7 to 1.7% of the dNTPs are turned over to dNMPs during replication compared with 6 to 13% for Pol III. The error rates measured for Pol I under conditions used for oligodeoxynucleotide-directed mutagenesis are sufficiently low that extraneous errors should not be induced when the concentrations of dNTPs are balanced. However, even higher fidelity will be obtained using the lowest concentrations of dNTPs consistent with efficient replication (~20 μ m ). Highly unbalanced concentrations as used in pulsed labelling should be avoided.

Electron micrographs of RecA-DNA filaments, formed under several different conditions, have been analyzed and the filament images reconstructed in three dimensions. In the presence of ATP and a non-hydrolyzable ATP analog. ATP-gamma-S, the RecA protein forms with DNA a right-handed helical complex with a pitch of approximately 95 A. The most detailed view of the filament was obtained from analysis of RecA filaments on double-stranded DNA in the presence of ATP-gamma-S. There are approximately six subunits of RecA per turn of the helix, but both this number and the pitch are variable. From the examination of single filaments and filament-filament interactions, a picture of an extremely flexible protein structure emerges. The subunits of RecA protein are seen to be arranged in such a manner that the bound DNA must be partially exposed and able to come into contact with external DNA molecules. The RecA structure determined in the presence of ATP-gamma-S appears to be the same as the "pre-synaptic" state that occurs with ATP, in which there is recognition and pairing between homologous DNA molecules.

The effect of hairpin (cruciform) size on the regulation of gene expression was investigated by cloning a series of palindromic sequences into the non-essential J-F intercistronic region of the bacteriophage φX174 ins6 genome. Genetic stability of the insert sequence and its effect on the growth efficiency of the phage was used as an initial measure of the biological consequence of hairpin insertions. Multimers of increasing size of the BamHI linker sequence C-C-G-G-A-T-C-C-G-G were inserted into the PvuII site of the parental strain ins6. The largest hairpin that could be constructed and maintained in the φX174 genome had a stem length of 22 base-pairs and a loop size of four nucleotides (linker tetramer). However, this structure proved to be disadvantageous to the phage and was rapidly deleted from its genome. Trimer inserts were more stable, but were eventually deleted also. Monomer and dimer inserts, though genetically stable, decreased the growth efficiency of the phage as judged by competitive growth experiments and measurements of burst size. The physical formation of these hairpins was shown by restriction digests of single-stranded DNA with BamHI and HpaII. We argue that these secondary structures form in vivo, at least in the single-stranded genome and the polycistronic mRNAs, and were responsible for the observed growth defects.

The A and A* proteins of phage phi X174 are encoded in the same reading frame in the viral genome; the smaller A protein is the result of a translational start signal with the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the phi X 174 A* gene, previously cloned into pCQV2 under lambda repressor control, into a TAG stop codon. The altered A gene was then inserted back into phi X replicative form DNA to produce an amber mutant, phi XamA*. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su+ cells infected with phi XamA*, phi X gene A protein, altered at one amino acid, was synthesized at normal levels; A* protein was not detectable. These observations indicate that the A* protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of phi X174 DNA or the packaging of the viral strand under the conditions tested.

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. We have suggested that RecA might participate directly in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g. pyrimidine dimer) because of its partially unwound nature; DNA polymerase III will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site more often but with reduced fidelity. In support of this proposal, we have found that the phenotype of wild-type and mutant RecA for mutagenesis correlates with capacity to bind to double-stranded DNA. Wild-type RecA binds more efficiently to ultraviolet (u.v.)-irradiated, duplex DNA than to non-irradiated DNA. The RecA441 (Tif) protein that is constitutive for mutagenesis binds extremely well to double-stranded DNA with no lesions, whereas the RecA430 protein that is defective in mutagenesis binds poorly even to u.v.-irradiated DNA. The RecA phenotype also correlates with capacity to use duplex DNA as a cofactor for cleavage of the LexA repressor protein for SOS-controlled operons. Wild-type RecA provides efficient cleavage of LexA only with u.v.-irradiated duplex DNA; RecA441 cleaves well with non-irradiated DNA; RecA430 gives very poor cleavage even with u.v.-irradiated DNA. We conclude that the interaction of RecA with damaged double-stranded DNA is likely to be a critical component of SOS mutagenesis and to define a pathway for the LexA cleavage reaction as well.

Binding of Escherichia coli RNA polymerase to φX174 DNA replicative form (RF) has been studied by electron microscopy. Samples of the binary complexes were spread for observation upon polylysine-coated carbon films. Binding was obtained with both RFI and RFIII forms of the DNA; complexes formed with the former were treated with restriction enzyme Pst I before spreading. A histogram constructed from the positions of 558 polymerase molecules bound to 181 DNA strands exhibited three prominent, sharp peaks at 3.3 × 10 2 nucleotides, 39.7 × 10 2 nucleotides and 49.0 × 10 2 nucleotides from the Pst I cleavage point. These positions correspond closely to those of the D, A and B promoter sequences, as derived from φX174 DNA sequence data by Sanger et al. (1977).

Electron microscope partial denaturation maps of two viral DNAs, simian virus 40 and φX174 replicative form, have been obtained. A simple computer program has been developed to predict denaturation maps from any given DNA sequence, based on the percentage of A · T base-pairs along the molecule. Maps constructed from the SV40 DNA and φX174 replicative form DNA base sequence show a good correlation with the experimental maps. The results show that the regions of a DNA molecule that denature first are, in fact, those regions with the highest content of adenine and thymine base-pairs.

The protein moiety of bacteriophage φX174 can be dissociated into subunits of uniform molecular size by treatment with sodium dodecyl sulfate at elevated temperature and pH. Subsequent separation of protein from phage DNA may be effected by ammonium sulfate fractionation. About 60% of the protein of the starting material is recovered by this procedure. The physical state of the subunit is markedly dependent upon solvent conditions. On removal of detergent or lowering of pH the subunits aggregate. These aggregates may be dispersed by addition of detergent in alkaline (pH 10·5) solution.

The course of virus protein synthesis in E. coli C infected with bacteriophage φ X174 has been determined by pulse labeling of infected cells with a radioactive amino acid during the course of virus development followed by immunological isolation of virus antigen from artificially disrupted cells. The results show that virus antigen synthesis is initiated about 8 min after infection and that the rate of synthesis reaches a maximum at about 15 min. Under our conditions, the maximum rate of synthesis constitutes only about 6 to 10% of the total protein synthesis in the infected cell and continues at this rate till lysis occurs. Studies on the growth of the host cell show that the rates of cell division and RNA, DNA and protein synthesis are virtually unchanged during the major portion of active virus synthesis. Two functionally different types of virus adsorption to the host cell can be distinguished.

The infective, intracellular form of bacteriophage φX174 DNA has been reported to be much more resistant to inactivation by u.v. irradiation than is" the infective single-stranded DNA isolated from mature virus. The tenfold smaller inactivation cross-section of the RFt is consistent with its postulated double-stranded structure (Sinsheimer, Starman, Nagler & Guthrie, 1962).

In shadowed electron micrographs, bacteriophages φX174 and S13 appear to consist of twelve “knobs” arranged as at the vertices of an icosahedron. The existence of these “knobs” is confirmed by the appearance of both positively and negatively stained preparations of φX174. On heating or treatment with concentrated acetate or urea, φX174 extrudes strands up to 1·3μ in length. The strand diameter may range from less than 10 A to 40 A or above. The strands are disrupted by DNase or by heating to 80°C. They are believed to consist of DNA, irregularly coated by protein. Purified φX174 DNA, like other single-stranded nucleic acids, appears as inhomogeneous strands or “random coils” in electron micrographs. A model is proposed for the structure of φX174 in which the bulk of the phage consists of deoxyribonucleoprotein. The configuration of the DNA is thus maintained by direct combination with about 80% of the total protein. The remainder is divided into sixty identical units which are arranged in groups of five to form the twelve “knobs” of the icosahedrally symmetrical outer protein shell.