In vitro replication of adeno-associated virus DNA: enhancement by extracts from adenovirus-infected HeLa cells.

Previously we have described an in vitro assay for the replication of adeno-associated virus type 2 (AAV2) DNA. Addition of the AAV2 nonstructural protein Rep68 to an extract from uninfected cells supports the replication of linear duplex AAV DNA. In this report, we examine replication of linear duplex AAV DNA in extracts from either uninfected or adenovirus (Ad)-infected HeLa cells. The incorporation of radiolabeled nucleotides into full-length linear AAV DNA is 50-fold greater in extracts from Ad-infected cells than in extracts from uninfected cells. In addition, the majority of the labeled full-length AAV DNA molecules synthesized in the Ad-infected extract have two newly replicated strands, whereas the majority of labeled full-length AAV DNA molecules synthesized in the uninfected extract have only one newly replicated strand. The numbers of replication initiations on original templates in the two assays are approximately the same; however, replication in the case of the Ad-infected cell extract is much more likely to result in the synthesis of a full-length AAV DNA molecule. Most of the newly replicated molecules in the assay using uninfected cell extracts are in the form of stem-loop structures. We hypothesize that Ad infection provides a helper function related to elongation during replication by a single-strand displacement mechanism. In the assay using the uninfected HeLa cell extract, replication frequently stalls before reaching the end of the genome, causing the newly synthesized strand to be displaced from the template, with a consequent folding on itself and replication back through the inverted terminal repeat, using itself as a template. In support of this conjecture, replication in the uninfected cell extract of shorter substrate molecules is more efficient, as measured by incorporation of radiolabeled nucleotides into full-length substrate DNA. In addition, when shorter substrate molecules are used as the template in the uninfected HeLa cell assay, a greater proportion of the labeled full-length substrate molecules contain two newly replicated strands. Shorter substrate molecules have no replicative advantage over full-length substrate molecules in the assay using an extract from Ad-infected cells.     

Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro.

Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers. Thus, similarly to its activity on UV-irradiated single-stranded DNA, DNA polymerase III holenzyme can bypass pyrimidine photodimers in the more complex replicative form --->single-strand replication, which involves, in addition to the polymerizing activity, the unwinding of the duplex by the rep helicase and the participation of a more complex multiprotein replisome.     

A rapid DNA assembling strategy mediated by direct full-length polymerase chain reaction

An efficient DNA assembling strategy was developed here modified from Class-IIS endonuclease mediated DNA splicing by directed ligation (SDL). Benefited from the full-length PCR directly using ligation products as template, this strategy required less effort and less time to obtain the assembled full-length DNA. The advantages of this strategy made it a rapid and easy-to-perform gene splicing and multiple site-directed mutagenesis approach especially practicable when more fragments need to be assembled at the same time.     

Displacement synthesis of globin complementary DNA: evidence for sequence amplification

We have examined a cDNA displacement synthesis procedure in which the extent of precursor incorporation and the unusual kinetics of displacement synthesis suggest a unique replicative form of DNA and the occurrence of multiple rounds of displacement synthesis, leading to amplification of mRNA sequences. Globin double-stranded DNA containing a hairpin loop was extended by the addition of a homopolymer to the 3' end. This was followed by displacement synthesis with the Klenow fragment of DNA polymerase I that was primed by an oligonucleotide hybridized to the homopolymer. Thus, the hairpin cDNA was copied to form an open duplex with an inverted repetition of globin sequences. These molecules can then serve as templates for additional synthesis which would be primed from oligomers bound the homopolymer. Globin cDNA sequences appear to be amplified 10-fold or more by this procedure. Globin cDNA obtained by displacement synthesis was similar in size to the original template. However, displaced molecules associate to the extent that they are not readily resolved by electrophoresis or sedimentation under nondenaturing conditions. Restriction endonuclease digests of 32P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S1 nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.     

Random mutagenesis and recombination of sam1 gene by integrating error-prone PCR with staggered extension process

An efficient method for creating a DNA library is presented in which gene mutagenesis and recombination can be introduced by integrating error-prone PCR with a staggered extension process in one test tube. In this process, less than 15 cycles of error-prone PCR are used to introduce random mutations. After precipitated and washed with ethanol solution, the error-prone PCR product is directly used both as template and primers in the following staggered extension process to introduce DNA recombination. The method was validated by using adenosyl-methionine (AdoMet) synthetase gene, sam1, as a model. The full-length target DNA fragment was available after a single round. After being selected with a competitive inhibitor, ethionine, a mutated gene was obtained that increased AdoMet accumulation in vivo by 56%.     

Titles of related papers in other sections

We have examined a cDNA displacement synthesis procedure in which the extent of precursor incorporation and the unusual kinetics of displacement synthesis suggest a unique replicative form of DNA and the occurrence of multiple rounds of displacement synthesis, leading to amplification of mRNA sequences. Globin double-stranded DNA containing a hairpin loop was extended by the addition of a homopolymer to the 3′ end. This was followed by displacement synthesis with the Klenow fragment of DNA polymerase I that was primed by an oligonucleotide hybridized to the homopolymer. Thus, the hairpin cDNA was copied to form an open duplex with an inverted repetition of globin sequences. These molecules can then serve as templates for additional synthesis which would be primed from oligomers bound the homopolymer. Globin cDNA sequences appear to be amplified 10-fold or more by this procedure. Globin cDNA obtained by displacement synthesis was similar in size to the original template. However, displaced molecules associate to the extent that they are not readily resolved by electrophoresis or sedimentation under nondenaturing conditions. Restriction endonuclease digests of ³²P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S₁ nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.     

Replication of the linear mitochondrial DNA of Tetrahymena pyriformis.

1. Electron micrographs of the linear mtDNA from Tetrahymena pyriformis strain GL show linear molecules with a duplex 'eye' of variable size in the middle. This indicates that replication of this DNA starts near the middle of the molecule and proceeds bidirectionally to the ends, as previously shown for the mtDNA of strain ST (Arnberg, A.C., Van Bruggen, E.F.J., Clegg, R.A., Upholt, W.B. and Borst, P. (1974) Biochim. Biophys. Acta 361, 266-276). The mtDNAs of these two strains have little base sequence homology beyond the ribosomal RNA cistron (Goldbach, R.W., Bollen-De Boer, J.E., Van Bruggen, E.F.J. and Borst, P. (1978) Biochim. Biophys. Acta 521, 187-197). 2. Electron micrographs of mtDNA from strain ST, spread under non-denaturing conditions, contain only molecules with fully duplex ends. mtDNA spread under conditions of early denaturation contains duplex loops on one end (40% of all molecules) or both ends (37%). The loops are stable to partial denaturation and vary in size from 0.15 to approximately 1.0 micron, most loops measuring 0.25--0.40 micron. No loops are formed with single-stranded DNA under analogous conditions and we conclude from this result that loop formation is based on the presence of straight, rather than inverted, duplications near the ends. 3. When full-length 3H-labelled mtDNA from strain ST, 32P-labelled at the 5'-termini with T4 polynucleotide kinase, was sedimented in alkaline sucrose gradients, greater than 70% of the 3H and less than 30% of the 32P cosedimented with full-length molecules; the remaining 32P sedimented heterogeneously and predominantly with the DNA less than 10% the size of intact single strands. Brief incubations of full-length mtDNA with DNA polymerase I from Escherichia coli and labelled dNTPs at 15 degrees C did not lead to preferential labelling of terminal EcoRI fragments of the DNA. From these results we infer that the DNA contains nicks or gaps near the termini and that these are not bordered by free 3'-OH groups. 4. A model is presented in which straight sequence repetitions at the termini of Tetrahymena pyriformis mtDNA are involved in the later stages of replication. This model can also account for the pronounced terminal heterogeneity previously observed in this DNA.     

Sequence requirements for protein-primed DNA replication of bacteriophage PRD1

In vitro studies have demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism. No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA. We have determined the minimal origin of replication by analyzing the template activity of various deletion derivatives. Our results showed that the terminal 20 base-pairs of ITR are required for efficient in vitro DNA replication. We have found that, within the minimal replication origin region, there are complementary sequences. A site-specific mutagenesis analysis showed that most of the point mutations in the complementary sequences markedly reduced the template activity. The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3'-terminal C residue is required for the initiation of PRD1 DNA replication in vitro.     

Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

The RNA world hypothesis states that the early evolution of life went through a stage where RNA served as genome and as catalyst. The replication of RNA world organisms would have been facilitated by ribozymes that catalyze RNA polymerization. To recapitulate an RNA world in the laboratory, a series of RNA polymerase ribozymes was developed previously. However, these ribozymes have a polymerization efficiency that is too low for self-replication, and the most efficient ribozymes prefer one specific template sequence. The limiting factor for polymerization efficiency is the weak sequence-independent binding to its primer/template substrate. Most of the known polymerase ribozymes bind an RNA heptanucleotide to form the P2 duplex on the ribozyme. By modifying this heptanucleotide, we were able to significantly increase polymerization efficiency. Truncations at the 3'-terminus of this heptanucleotide increased full-length primer extension by 10-fold, on a specific template sequence. In contrast, polymerization on several different template sequences was improved dramatically by replacing the RNA heptanucleotide with DNA oligomers containing randomized sequences of 15 nt. The presence of G and T in the random sequences was sufficient for this effect, with an optimal composition of 60% G and 40% T. Our results indicate that these DNA sequences function by establishing many weak and nonspecific base-pairing interactions to the single-stranded portion of the template. Such low-specificity interactions could have had important functions in an RNA world.     

Discrimination between translesion synthesis and template switching during bypass replication of thymine dimers in duplex DNA

The goal of this study was to determine whether bypass replication occurs by translesion synthesis or template switching (copy choice) when a duplex molecule carrying a single cis,syn-cyclobutane thymine dimer is replicated in vitro by human cell extracts. Circular heteroduplex DNA molecules were constructed to contain the SV40 origin of replication and a mismatch opposite to or nearby the dimer. Control molecules with only the mismatch were also prepared. Heteroduplexes were methylated at CpG islands and replicated in vitro (30 min). Following bisulfite treatment, the nascent DNA complementary to the dimer-containing template was distinguished from the other three strands by methylation-specific polymerase chain reaction. Cloning and sequencing of polymerase chain reaction products revealed that 80-98% carried the sequence predicted for translesion synthesis, with two adenines incorporated opposite the dimer. The fraction of clones with sequence predictive of template switching was reduced when extracts deficient in mismatch repair or nucleotide excision repair activities were used to replicate the heteroduplex molecules. These results support the conclusion that lesion bypass during in vitro replication of duplex DNA containing thymine dimers occurs by translesion synthesis.     

Characterization of the bacteriophage T4 gene 41 DNA helicase

The T4 gene 41 protein and the gene 61 protein function together as a primase-helicase within the seven protein bacteriophage T4 multienzyme complex that replicates duplex DNA in vitro. We have previously shown that the 41 protein is a 5' to 3' helicase that requires a single-stranded region on the 5' side of the duplex to be unwound and is stimulated by the 61 protein (Venkatesan, M., Silver L. L., and Nossal, N. G. (1982) J. biol. Chem. 257, 12426-12434). The 41 protein, in turn, is required for pentamer primer synthesis by the 61 protein. We now show that the 41 protein helicase unwinds a partially duplex DNA molecule containing a performed fork more efficiently than a DNA molecule without a fork. Optimal helicase activity requires greater than 29 nucleotides of single-stranded DNA on the 3' side of the duplex (analogous to the leading strand template). This result suggests the 41 protein helicase interacts with the leading strand template as well as the lagging strand template as it unwinds the duplex region at the replication fork. As the single-stranded DNA on the 3' side of a short duplex (51 base pairs) is lengthened, the stimulation of the 41 protein helicase by the 61 protein is diminished. However, both the 61 protein and a preformed fork are essential for efficient unwinding of longer duplex regions (650 base pairs). These findings suggest that the 61 protein promotes both the initial unwinding of the duplex to form a fork and subsequent unwinding of longer duplexes by the 41 protein. A stable protein-DNA complex, detected by a gel mobility shift of phi X174 single-stranded DNA, requires both the 41 and 61 proteins and a rNTP (preferably rATP or rGTP, the nucleotides with the greatest effect on the helicase activity). In the accompanying paper, we report the altered properties of a proteolytic fragment of the 41 protein helicase and its effect on in vitro DNA synthesis in the T4 multienzyme replication system.     

Bleomycin fragmentation of duplex DNA occurs as staggered single-strand scissions.

Electron microscopy of purified full-length linear duplex molecules produced by bleomycin reaction with PM2 DNA revealed low frequencies of closed circular duplex molecules as well as linear duplex molecules with opposed ends (cyclized molecules which have dissociated to yield a gap between the termini). The occurrence of these latter forms indicates that double-strand scissions produced by bleomycin reaction consist of two single-strand scissions which are physically staggered on the complementary strands. Analysis of the temperature dependence for cyclization led to the estimate that an average of 1.7 +/- 0.44 base-pairs (2.6 +/- 0.5 base pairs without base-stacking energies) occur between the staggered breaks. The reassociated termini cannot be ligated with T4 ligase. When PM2 DNA was fragmented at several sites within each molecule, circular duplexes and linear duplexes with opposed ends with a range of sizes from 350 base pairs up to full-length PM2 DNA were observed. Analysis of the frequency distribution of lengths of these fragments indicates that most, if not all, of the specific sites for bleomycin-directed double-strand scissions in PM2 DNA contain representatives of the same two base single-stranded termini.     

Demonstration of two alpha-globin genes per human haploid genome for normals and Hb J Mexico.

Complementary DNA (cDNA) was prepared with viral RNA-dependent DNA polymerase using human globin messenger RNA (mRNA) as template. By selective hydridization to globin mRNA from beta-thalassaemics a probe which was greater than 85% complementary to alpha-globin mRNA was purified. This was hybridized in cDNA excess to human genomic DNA, and the rate and extent of hybridization confirmed that there are two genes for alpha-globin per haploid genome. Cellular DNA was also prepared from peripheral blood from cases expressing the alpha-globin chain mutant Hb J Mexico to varying extents. This DNA was identical in hybridization behaviour to normal DNA demonstrating that the imbalanced mutant chain synthesis seen physiologically is not due to a gene deletion.     

Control of reaction chemoselectivity with a circular DNA template in the ligation of short oligodeoxyribonucleotides.

A study has been conducted to examine the chemoselective control attained in the ligation of short oligodeoxyribonucleotides (ODNs) directed by association on a circular DNA template through triplex formation relative to the same ligation on a single-strand DNA template through duplex formation. The highly efficient, chemospecific ligation achieved under a variety of conditions on the circular DNA template presumably arises from the higher substrate-template association resulting in improved transition state accessibility and protection of the ODNs from side reactions.