Differential Activities of DNA Polymerases in Processing Ribonucleotides during DNA Synthesis in Archaea

Consistent with the fact that ribonucleotides (rNTPs) are in excess over deoxyribonucleotides (dNTPs) in vivo, recent findings indicate that replicative DNA polymerases (DNA Pols) are able to insert ribonucleotides (rNMPs) during DNA synthesis, raising crucial questions about the fidelity of DNA replication in both Bacteria and Eukarya. Here, we report that the level of rNTPs is 20-fold higher than that of dNTPs in Pyrococcus abyssi cells. Using dNTP and rNTP concentrations present in vivo, we recorded rNMP incorporation in a template-specific manner during in vitro synthesis, with the family-D DNA Pol (PolD) having the highest propensity compared with the family-B DNA Pol and the p41/p46 complex. We also showed that ribonucleotides accumulate at a relatively high frequency in the genome of wild-type Thermococcales cells, and this frequency significantly increases upon deletion of RNase HII, the major enzyme responsible for the removal of RNA from DNA. Because ribonucleotides remain in genomic DNA, we then analyzed the effects on polymerization activities by the three DNA Pols. Depending on the identity of the base and the sequence context, all three DNA Pols bypass rNMP-containing DNA templates with variable efficiency and nucleotide (mis)incorporation ability. Unexpectedly, we found that PolD correctly base-paired a single ribonucleotide opposite rNMP-containing DNA templates. An evolutionary scenario is discussed concerning rNMP incorporation into DNA and genome stability.     

Cloning of a functional replication origin of phage G4 into the genome of phage M13.

A chimeric single-stranded DNA phage, M13Gori1, has been formed as a result of the in vitro insertion of a 2216 base-pair HaeII fragment of bacteriophage G4 replicative form DNA into the replicative form DNA of bacteriophage M13. The inserted G4 DNA carries the dnaG-dependent origin for G4 complementary strand synthesis. The cloned G4 origin functions both in vivo and in vitro in the conversion of M13Gori1 single-stranded viral DNA to the duplex replicative form by a rifampicin-resistant mechanism. Labelling of the 3′ terminus of the single discontinuity in M13Gori1 replicative form II molecules synthesized in crude extracts and subsequent restriction analysis indicate that M13Gori1 complementary strand synthesis can be initiated at either the RNA polymeraseprimed M13 origin or at the dnaG-primed G4 origin. The M13Gori1 complementary strand initiated at the G4 origin terminates in the vicinity of the G4 origin after progressing around the circular template and traversing the M13 origin region, indicating the absence of a specific nucleotide sequence in the M13 origin for termination of the newly formed complementary strand. The ability of this chimeric phage to utilize the cloned G4 origin in vivo even in the presence of the presumed M13 pilot protein (gene 3 protein) indicate that the nucleotide sequence of the replication origin is sufficient for recognizing the appropriate initiation enzymes. Since decapsidation of M13 is tightly coupled to replicative form formation, initiation at the G4 origin, located over 1000 nucleotides from the M13 complementary strand origin, indicates that widely separated nucleotide sequences contained in the filamentous virion can be exposed to the cell cytoplasm during eclipse.     

Bacteriophage φX174 and G4 RF DNA replicative intermediates: A comparative study using different isolation procedures

We have isolated replicative intermediates of bacteriophage φX174 and the related baeteriophage G4, during RF (replicative form) DNA replication using different procedures. Biochemical and electron microscopic analysis of φX and G4 DNA replicative intermediates isolated by the same procedure, showed no significant differences. In the replication cycle of both phages rolling circles and gapped RF DNA molecules are the predominant replicative intermediates. It is concluded that G4 RF DNA also replicates according to a rolling circle model and not according to a D-looped replication model as proposed by Godson (1977b).     

Role of genes O and P in the replication of bacteriophage lambda DNA.

DNA replication in coliphage λ occurs in two stages. The first round of replication generates mainly circular progeny DNA by a double-branched θ-type replicative form (Ogawa et al., 1968; Schnös &; Inman, 1970). In the late stage of λ DNA replication, however, σ-type rolling-circle replicative form DNA molecules, which produce multigenomic linear concatemers, are primarily found (Takahashi, 1974).At both early and later times, a temperature shift of λ Ots or Pts infected cells from 32 °C (permissive) to 43 °C (non-permissive temperature) caused a rapid reduction of the rate of radioactive precursor incorporation into λ DNA, showing that the gene O and P products are essential for the continuation of λ DNA synthesis. Observations on the molecular fine structure of the replicating fork after a temperature shift revealed characteristic long “single-strand connections” and single-strand “whiskers” at the branch point. These observations suggest that λ gene O and P products are directly involved in the propagation of daughter strands.     

DNA synthesis in nucleotide-permeable Escherichia coli cells. VII. Conversion of phi chi-174 DNA to its replicative form

On incubation with deoxynucleoside triphosphates and rATP, ether-treated (nucleotide-permeable) cells convert the single-stranded DNA of adsorbed bacteriophage φX174 particles to the double-stranded replicative forms. The main final product is the doubly-closed replicative form, RFI; a minor product is the relaxed form II. Interruptions in the nascent complementary strand of the viral DNA result in pieces corresponding to 5 to 10% of the unit length of the viral DNA. Pieces of similar size were previously seen in studies of the replication synthesis of Escherichia, coli DNA in ether-treated cells. Since the conversion of the single-stranded φX174 DNA to replicative form is known to be mediated entirely by host factors, it is argued that the viral single strands are replicated by macromolecular factors involed in the replication of E. coli DNA and that this is the reason why new φX174 DNA appears in short pieces. Possible consequences of this interpretation for an understanding of duplex replication are discussed. The joining of the short pieces of complementary φX174 DNA is inhibited at low deoxynucleoside triphosphate concentration (1 μM) but not by nicotinamide mononucleotide, which inhibits the NAD-dependent DNA ligase and blocks the conversion of RFII to RFI in ether-treated cells. The results are discussed with respect to previous studies on cell-DNA synthesis (Geider, 1972). It is argued that there are two polynucleotide joining mechanisms, of which only one requires NAD-dependent ligase action.     

A role for single-strand breaks in bacteriophage phi-X174 genetic recombination

Formation of genetic recombinants in bacteriophage φX174 is stimulated up to 50-fold in host cells carrying the recA⁺ allele by subjecting the virus particles to ultraviolet irradiation before infection, or by starving the host cell for thymine during infection; in recA host strains no such increases are observed.φX174 replicative form DNA molecules formed in vivo from ultraviolet-irradiated bacteriophage consist of an intact, circular full-length viral (+) strand and a partially complete complementary (?) strand extending from the point of origin of complementary strand DNA synthesis to an ultraviolet lesion. φX174 replicative form DNA molecules formed in thymine-deficient host strains during thymine starvation have nearly complete circular viral (+) and complementary (?) strands, which contain random single-strand nicks or gaps.Correlation of these structures with the observed increases in recombination suggests that single-strand “breaks” are aggressive intermediate structures in the formation of φX174 genetic recombinants mediated by the host recA⁺ gene product.     

Cat fleas (Ctenocephalides felis) carrying Rickettsia felis and Bartonella species in Hong Kong

Fleas are commonly recorded on stray as well as domestic dogs and cats in Hong Kong. Fleas can be a major cause of pruritus in dogs and cats and also vectors of potentially zoonotic bacteria in the genera Rickettsia and Bartonella. Morphological examination of 174 fleas from dogs and cats living in Hong Kong revealed only cat fleas (Ctenocephalides felis). Cytochrome c oxidase subunit 1 gene (cox1) genotyping of 20 randomly selected specimens, revealed three cox1 haplotypes (HK-h1 to HK-h3). The most common haplotype was HK-h1 with 17 specimens (17/20, 85%). HK-h1 was identical to cox1 sequences of fleas in Thailand and Fiji. HK-h1 and HK-h2 form a distinct cat flea cox1 clade previously recognized as the Clade 3. HK-h3 forms a new Clade 6. A multiplex Bartonella and Rickettsia real-time PCR of DNA from 20 C. felis found Bartonella and Rickettsia DNA in three (15%) and ten (50%) C. felis, respectively. DNA sequencing confirmed the presence of R. felis, B. clarridgeiae and Bartonella henselae. This is the first reported study of that kind in Hong Kong, and further work is required to expand the survey of companion animals in the geographical region. The sampling of fleas on domestic cats and dogs in Hong Kong revealed them to be exclusively infested by the cat flea and to be harbouring pathogens of zoonotic potential.     

Structure and primase-mediated activation of a bacterial dodecameric replicative helicase

Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.     

Molecular Interplay between the Replicative Helicase DnaC and Its Loader Protein DnaI from Geobacillus kaustophilus

Helicase loading factors are thought to transfer the hexameric ring-shaped helicases onto the replication fork during DNA replication. However, the mechanism of helicase transfer onto DNA remains unclear. In Bacillus subtilis, the protein DnaI, which belongs to the AAA+ family of ATPases, is responsible for delivering the hexameric helicase DnaC onto DNA. Here we investigated the interaction between DnaC and DnaI from Geobacillus kaustophilus HTA426 (GkDnaC and GkDnaI, respectively) and determined that GkDnaI forms a stable complex with GkDnaC with an apparent stoichiometry of GkDnaC₆-GkDnaI₆ in the absence of ATP. Surface plasmon resonance analysis indicated that GkDnaI facilitates loading of GkDnaC onto single-stranded DNA (ssDNA) and supports complex formation with ssDNA in the presence of ATP. Additionally, the GkDnaI C-terminal AAA+ domain alone could bind ssDNA, and binding was modulated by nucleotides. We also determined the crystal structure of the C-terminal AAA+ domain of GkDnaI in complex with ADP at 2.5 Å resolution. The structure not only delineates the binding of ADP in the expected Walker A and B motifs but also reveals a positively charged region that may be involved in ssDNA binding. These findings provide insight into the mechanism of replicative helicase loading onto ssDNA.     

Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target.     

Replication of Coliphage M-13 II. Intracellular Deoxyribonucleic Acid Forms Associated with M-13 Infection of Mitomycin C-treated Cells

Intracellular deoxyribonucleic acid (DNA) forms associated with bacteriophage M-13 infection have been isolated and characterized. Escherichia coli HF4704 (F⁺, hcr⁻, thy⁻) cells were treated with mitomycin C to inhibit host-cell DNA synthesis and were then infected with phage M-13. This treatment permitted radioactive labeling of phage-specific DNA forms with ³H-thymine. These labeled DNA components were characterized by sucrose density sedimentation and equilibrium density gradient centrifugation in neutral and ethidium bromide CsCl gradient. Two double-stranded circular forms were found with properties analogous to the replicative form I and replicative form II of X174. A third component, identified as single-stranded DNA, was isolated in some samples removed 45 min after phage synthesis was initiated.     

Figure-8 configuration of dimers of S13 and φX174 replicative form DNA

Evidence from electron microscopy of the replicative form of S13 and φX174 DNA shows the presence of a “figure-8” configuration. This species consists of two monomer length and one dimer length circular strands in covalently closed circular form and containing a fused junction that divides the molecule into two equal circular segments. Its existence is supported by the demonstration that it is converted by digestion with the restriction endonuclease of Hemophilus influenzae strain Rd to α- and X-shaped forms that retain the fused junction, and by examination by electron microscopy in the presence of ethidium bromide, which eliminates tangling and accidental overlays of parts of the DNA molecules. Kgure-8s are present to the extent of about 5% of the dimers present in replicative form DNA. They are proposed to be intermediates in genetic recombination in S13 and φX174.     

Extrachromosomal DNA in Bacillus thuringiensis var. kurstaki, var. finitimus, var. sotto, and in Bacillus popilliae

Four entomopathogenic bacteria contained extrachromosomal deoxyribonucleic acid (DNA) molecules of various sizes. Bacillus thuringiensis var. kurstaki contained twelve elements banding on agarose gels that ranged from 0.74 to > 50 × 10⁶ daltons, three of which were giant extrachromosomal DNA elements. B. thuringiensis var. sotto contained one giant extrachromosomal DNA element with a molecular size of about 23.5 × 10⁶ daltons and two lesser elements of 0.80 and 0.62 × 10⁶ daltons. B. thuringiensis var. finitimus harbored two giant DNA elements corresponding to >50 × 10⁶ daltons and two lesser bands with relative small size (0.98 and 0.97 × 10⁶ daltons). B. popilliae contained no giant extrachromosomal DNA elements but did contain two smaller elements corresponding to 4.45 and 0.58 × 10⁶ daltons. The possible use of extrachromosomal DNA elements that prove to be autonomous replicons for recombinant DNA studies is discussed.     

Stimulation of polyoma DNA replication in isolated nucleoprotein complexes by factors from Drosophila embryos

Nucleoprotein complexes containing both form 1 and replicative intermediates of polyoma DNA prepared from nuclei of virus-infected mouse fibroblasts retain a limited ability to elongate progeny strands of the replicative intermediates. Compared to isolated nuclei, both the rate and the extent of strand elongation is greatly decreased. The isolated complexes synthesize initiator RNA and start new Okazaki fragments, but are deficient in the joining of these fragments. Addition of small amounts of an extract from 16 hours old Drosophila embryos corrects the deficiencies. The stimulatory activity of the extract can be partially purified and has been separated into two fractions by chromatography on Sepharose 6B. With immunological techniques we demonstrate that the mouse DNA polymerase-α, tightly bound to the complexes, is responsible for DNA strand elongation.The Drosophila α-polymerase present in one of the two fractions purified on Sepharose 6B cannot substitute for the mouse enzyme. The stimulatory activity of the Drosophila fractions is thus not due to α-polymerase.     

Mechanism of replication of phichi174 single-stranded DNA. IV. The parental viral strand is not conserved in the replicating DNA structure

The role of the infecting viral strand in the replication of bacteriophage φX174 replicative form DNA was studied by [³H]thymidine pulse-labeling Escherichia coli cells infected with ²H¹⁵N density-labeled phage. The products of a round of semi-conservative replicative form replication (in light medium) do not contain the original heavy viral strand by 15 minutes after infection or later in the presence of chloramphenicol. Similar results were obtained at earlier times in the absence of chloramphenicol. We conclude that the parental viral strand need not be conserved in the replicating DNA structure in succeeding rounds of replication.