Persistence and replication of plasmid DNA microinjected into early embryos of Xenopus laevis

The persistence and replication of defined circular and linear plasmid DNA molecules microinjected into fertilized eggs of Xenopus laevis were analyzed. For all plasmids tested, a small fraction of microinjected circular molecules was replicated; however, the overall copy numbers of either free form I or form II molecules usually did not increase through blastulation. In contrast, extensive amplification of input DNA sequences was seen whenever the microinjected DNA was assembled into high molecular weight concatemers. Moreover, the appearance and subsequent replication of injected sequences in high molecular weight DNA were enhanced when linear (form III), rather than circular, molecules were microinjected. The injected form III DNA was rapidly converted into long linear concatemers. All possible orientations of monomeric molecules within the concatemers were observed although, on occasion, head-to-tail orientations were favored. Long linear concatemers were replicated very efficiently, irrespective of the sequence of the input DNA. Form I and form II DNA molecules were also formed in the embryo from microinjected form III DNA. A small fraction of these circular forms was replicated, although overall copy numbers did not increase significantly. Form III molecules that remained monomeric were not observed to be replicated at all within our limits of detection. In some batches of embryos, form I and form II DNA molecules were replicated to the extent that overall copy number increased. Even in these cases, however, the amplification of long linear concatemers of the input DNA sequences was more efficient.     

DNA ligase I from Xenopus laevis eggs.

We have purified the major DNA ligase from Xenopus laevis eggs and raised antibodies against it. Estimates from SDS PAGE indicate that this DNA ligase is a 180 kDa protein. This enzyme is similar to the mammalian type I DNA ligase which is presumed to be involved in DNA replication. We have also analysed DNA ligase activity during X. laevis early development. Unfertilized eggs contain the highest level of activity reflecting the requirement for a large amount of DNA replicative enzymes for the period of intense replication following fertilization. In contrast with previous studies on the amphibians axolotl and Pleurodeles, the major DNA ligase activity detected during X. laevis early development is catalysed by a single enzyme: DNA ligase I. And the presence of this DNA ligase I in Xenopus egg before fertilization clearly demonstrates that the exclusion process of two forms of DNA ligase does not occur during X. laevis early development.     

Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase

The DNA-dependent protein kinase (DNA-PK), consisting of Ku and the DNA-PK catalytic subunit (DNA-PKcs), and the DNA ligase IV-XRCC4 complex function together in the repair of DNA double-strand breaks by non-homologous end joining. These protein complexes are also required for the completion of V(D)J recombination events in immune cells. Here we demonstrate that the DNA ligase IV-XRCC4 complex binds specifically to the ends of duplex DNA molecules and can act as a bridging factor, linking together duplex DNA molecules with complementary but non-ligatable ends. Although the DNA end-binding protein Ku inhibited DNA joining by DNA ligase IV-XRCC4, it did not prevent this complex from binding to DNA. Instead, DNA ligase IV-XRCC4 and Ku bound simultaneously to the ends of duplex DNA molecules. DNA ligase IV-XRCC4 and DNA-PKcs also formed complexes at the ends of DNA molecules, but DNA-PKcs did not inhibit ligation. Interestingly, DNA-PKcs stimulated intermolecular ligation by DNA ligase IV-XRCC4. In the presence of DNA-PK, the majority of the joining events catalyzed by DNA ligase IV-XRCC4 were intermolecular because Ku inhibited intramolecular ligation, but DNA-PKcs still stimulated intramolecular ligation. We suggest that DNA-PKcs-containing complexes formed at DNA ends enhance the association of DNA ends via protein-protein interactions, thereby stimulating intermolecular ligation.     

Adenovirus E4 34k and E1b 55k oncoproteins target host DNA ligase IV for proteasomal degradation

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.     

Reinvestigation of DNA ligase I in axolotl and Pleurodeles development.

We have recently shown that the exclusion process causing the replacement of DNA ligases II by DNA ligase I in amphibian eggs after fertilization does not occur in the case of Xenopus laevis [Hardy, S., Aoufouchi, S., Thiebaud, P., and Prigent, C., (1991) Nucleic Acids Res. 19, 701-705]. Since this result is in contradiction with the situation reported in axolotl and Pleurodeles we decided to reinvestigate such results in both species. Three different approaches have been used: (1) the substrate specificity of DNA ligase I; (2) the DNA ligase-AMP adduct reaction and (3) the immunological detection using antibodies raised against the X.laevis DNA ligase I. Our results clearly demonstrate that DNA ligase I activity is associated with a single polypeptide which is present in oocyte, unfertilized egg and embryo of both amphibians. Therefore, the hypothesis of a change in DNA ligase forms, resulting from an expression of the DNA ligase I gene in axolotl and Pleurodeles early development must be rejected. We also show that, in contradiction with published data, the unfertilized sea urchin egg contains a DNA ligase activity able to join blunt ended DNA molecules.     

Concatemers in DNA replication: Electron microscopic studies of partially denatured intracellular lambda DNA

We have verified, by identification of individual molecules in the electron microscope, that λ DNA concatemers are long linear molecules containing repeats of the mature phage DNA sequence. The molecules are not made up of whole multiples of the length of mature λ DNA and do not seem to contain specific start or end points. The concatemers, comprising about 20% of the molecular forms extracted late in infection, can be found in the absence of genetic recombination and in the presence or absence of maturation defects. It may be concluded that concatemers are normal intermediates in the late stage of λ replication.     

Structure-specific binding of the proto-oncogene protein DEK to DNA

The ubiquitous proto-oncogene protein DEK has been found to be associated with chromatin during the entire cell cycle. It changes the topology of DNA in chromatin and protein-free DNA through the introduction of positive supercoils. The sequence and structure specificities of DEK–DNA interactions are not completely understood. The binding of DEK to DNA is not sequence specific, but we describe here that DEK has a clear preference for supercoiled and four-way junction DNA. In the presence of topoisomerase II, DEK stimulates intermolecular catenation of circular DNA molecules. DEK also increases the probability of intermolecular ligation of linear DNA molecules by DNA ligase. These binding properties qualify DEK as an architectural protein.     

Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).     

Preparation and isolation of covalently closed circular rDNA molecules from DNA of Xenopus laevis.

We describe a method leading to the formation of closed circles of rDNA starting from total DNA of Xenopus laevis. Linear DNA molecules were digested with exonuclease 3 and self-annealed. Open circles were enriched and covalently closed by the simultaneous use of polynucleotide kinase, DNA polymerase and polynucleotide ligase. Closed circles of rDNA1 were shown to be alkali-resistant, to have higher density than linear molecules in cesium chloride density gradients containing ethydium bromide, and to have the sedimentation constant expected for a single repeat unit of rDNA comprehensive of its spacer.     

Knockdown of DNA ligase IV/XRCC4 by RNA interference inhibits herpes simplex virus type I DNA replication

Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IV-deficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection.     

Shear-induced assembly of lambda-phage DNA

Recombinant DNA technology, which is based on the assembly of DNA fragments, forms the backbone of biological and biomedical research. Here we demonstrate that a uniform shear flow can induce and control the assembly of lambda-phage DNA molecules: increasing shear rates form integral DNA multimers of increasing molecular weight. Spontaneous assembly and grouping of end-blunted lambda-phage DNA molecules are negligible. It is suggested that shear-induced DNA assembly is caused by increasing the probability of contact between molecules and by stretching the molecules, which exposes the cohesive ends of the otherwise undeformed lambda-phage DNA molecules. We apply this principle to enhance the kinetics and extent of DNA concatenation in the presence of ligase. This novel approach to controlled DNA assembly could form the basis for improved approaches to gene-chip and recombinant DNA technologies and provide new insight into the rheology of associating polymers.     

Analysis of the mechanism of interaction of simian Ku protein with DNA.

Ku protein is a relatively abundant DNA-binding protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku'). It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one protein bound per 30 bp of DNA. We have studied the mechanism of interaction of Ku protein with DNA in vitro, using protein extracted from cultured monkey cells. We find that the precise structure of the DNA ends is not important for binding, as Ku protein can bind to hairpin loops and to mononucleosomes. Bound protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase. Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl. We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences. Thus, Ku protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this protein in the nucleus.     

Homologous pairing of DNA molecules promoted by a protein from Ustilago

A protein from mitotic cells of Ustilago maydis was purified on the basis of its ability to reanneal complementary single strands of DNA. The protein catalyzed the uptake of linear single strands by super-helical DNA, but only in reactions with homologous combinations of single-strand fragments and super-helical DNA from phages phi X174 and fd. No reaction occurred with heterologous combinations. The protein also efficiently paired circular single strands and linear duplex DNA molecules. The product was a joint molecule in which the circular single strand displaced one strand of the duplex. Efficient pairing depended upon ATP, and ATPase activity was found associated with the purified protein. ATP-dependent reannealing of complementary single strands was not detectable in the rec1 mutant of Ustilago, which is deranged in meiotic recombination, as complete tetrads are rare, and is defective in radiation-induced mitotic gene conversion.     

The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate

A DNA replication system was developed that could generate rolling-circle DNA molecules in vitro in amounts that permitted kinetic analyses of the movement of the replication forks. Two artificial primer-template DNA substrates were used to study DNA synthesis catalyzed by the DNA polymerase III holoenzyme in the presence of either the preprimosomal proteins (the primosomal proteins minus the DNA G primase) and the Escherichia coli single-stranded DNA binding protein or the DNA B helicase alone. Helicase activities have recently been demonstrated to be associated with the primosome, a mobile multiprotein priming apparatus that requires seven E. coli proteins (replication factor Y (protein n'), proteins n and n', and the products of the dnaB, dnaC, dnaG, and dnaT genes) for assembly, and with the DNA B protein. Consistent with a rolling-circle mechanism in which a helicase activity permitted extensive (-) strand DNA synthesis on a (+) single-stranded, circular DNA template, the major DNA products formed were multigenome-length, single-stranded, linear molecules. The replication forks assembled with either the preprimosome or the DNA B helicase moved at the same rate (approximately 730 nucleotides/s) at 30 degrees C and possessed apparent processivities in the range of 50,000-150,000 nucleotides. The single-stranded DNA binding protein was not required to maintain this high rate of movement in the case of leading strand DNA synthesis catalyzed by the DNA polymerase III holoenzyme and the DNA B helicase.     

Initial steps in Haemophilus influenzae transformation. Donor DNA binding in the com10 mutant

The com10 mutant of Haemophilus influenzae binds donor DNA reversibly, but is deficient in uptake. The DNA binding has all the characteristics of interaction with a protein receptor; it is saturable, reversible, and specific. However, binding specificity is 6-fold weaker in com10 than is uptake specificity in wild-type. The binding of small (120 base pairs) and large (14,400 base pairs) DNA molecules were compared. For small molecules, binding data fitted a straight line by Scatchard analysis (Bmax = 4.8 DNA molecules/cell, Kd = 0.5 X 10(-9) M). In contrast, for large DNA molecules, the Scatchard plot was not linear. A high affinity binding (Kd = 0.4 X 10(-12) M) and a lower affinity binding (Kd = 1.2 X 10(-11) M) were found with a total number of 3 molecules bound per cell. In wild-type cells, 3.2 large molecules were taken up per cell, whereas up to 40 small 120-base pair DNA fragments were taken up per cell. Uptake of small DNA molecules followed a Michaelis-Menten function with a Km of 0.5 X 10(-9) M and a maximal initial velocity of 1.5 molecules/cell/min at room temperature. For large DNA molecules, maximal initial velocity was approximately 2 molecules/cell/min at room temperature. The analysis of the binding and uptake data suggest to us that a receptor or a receptor complex is responsible for the uptake of either a single large DNA molecule or, successively, a number of small DNA molecules.     

Folded, concatenated genomes as replication intermediates of bacteriophage T7 DNA.

A complex form of bacteriophage T7 DNA, containing up to several hundred phage equivalents of DNA, arises during replication of T7. The complex was stable to treatment with ionic detergent, Pronase, and phenol. The complex form normally exists for only a short time, corresponding to the phase of rapid T7 DNA synthesis. It is then converted to shorter molecules, both concatemers and unit-size DNA. The complex was stable up to the temperature of denaturation of the bihelix. It consisted of a series of loops amanating from a dense central core, as shownby electron microscopy. The complex form is similar to the relaxed Escherichia coli folded chromosome ('nucleoid'). The loops contained an average of 0.7 to 0.8 phage equivalent of DNA. During infection by phage with an amber mutation in gene 3 (endonuclease), formation of the complex occurred normally, but its maturation to unit-size DNA blocked. Before treatment with phenol, the complex contained short fragments of newly replicated DNA. These were released as single-stranded pieces during phenol treatment. A pathway for T7 DNA replication is indicated in which the flow of material is from unit-size DNA to linear concatemers to the complex form, and then back to unit-size DNA by way of linear concatemers.     

Addition of oligonucleotides to the 5''-terminus of DNA by T4 RNA ligase.

Bacteriophage T4-induced RNA ligase catalyzes the controlled template-independent addition of RNA to the 5'-phosphoryl end of large DNA molecules. Restriction enzyme-generated fragments of Co1E1 DNA with completely basepaired or cohesive ends and linear single-stranded ?X174 viral DNA were all good substrates. DNA molecules from 10 to 6000 nucleotides long were quantitatively joined in an hour to a number of different RNA homopolymers. The most efficient of these was A(pA)5; I(pI)5 and C(pC)5 were also utilized while U(pU)5 was not. The optimum ribohomopolymer length was six nucleotides. Joining of ribohomopolymers between 10 and 20 nucleotides long occurred at approximately 1/2 the maximal rate and a trimer was the shortest substrate. Thus RNA ligase provides a method for generating extensions of predetermined length and base composition at the 5'-end of large DNA molecules that complements the available procedures for extending the 3'-hydroxyl terminus of DNA.     

Complete enzymatic synthesis of DNA containing the SV40 origin of replication

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.