Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.     

Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle amplification

Many techniques in molecular biology require the use of pure nucleic acids in general and circular DNA (plasmid or mitochondrial) in particular. We have developed a method to separate these circular molecules from a mixture containing different species of nucleic acids using rolling circle amplification (RCA). RCA of plasmid or genomic DNA using random hexamers and bacteriophage Phi29 DNA polymerase has become increasingly popular for the amplification of template DNA in DNA sequencing protocols. Recently, we reported that the mutant single-stranded DNA binding protein (SSB) from Thermus thermophilus (TthSSB) HB8 eliminates nonspecific DNA products in RCA reactions. We developed this method for separating circular nucleic acids from a mixture having different species of nucleic acids. Use of the mutant TthSSB resulted in an enhancement of plasmid or mitochondrial DNA content in the amplified product by approximately 500×. The use of mutant TthSSB not only promoted the amplification of circular target DNA over the background but also could be used to enhance the amplification of circular targets over linear targets.     

Construction of a circular single-stranded DNA template containing a defined lesion

We report a concise and efficient method to make a circular single-stranded DNA containing a defined DNA lesion. In this protocol, phagemid DNA containing Uracil is used as a template to synthesize a complementary DNA strand using T7 DNA polymerase and an oligonucleotide primer including a site-specific DNA lesion. The ligated lesion-containing strand can be recovered after the phage-derived template DNA is degraded by treatment with E. coli Uracil DNA glycosylase and Exonucleases I and III. The resulting product is a circular single-stranded DNA containing a defined DNA lesion suitable for in vitro translesion replication assays.     

Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.     

TempliPhi,phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing

We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.     

Synthesis of single-stranded plasmid pT181 DNA in vitro. Initiation and termination of DNA replication

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. The free 3'-hydroxyl end at the nick is presumably used as primer for leading strand DNA synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. Recombinant plasmids were constructed that contain two pT181 origins of replication in either direct or inverted orientation. In vitro replication of the plasmid carrying two origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These results suggest that RepC can reinitiate replication at the second origin by nicking partially replicated, relaxed DNA. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.     

Bypass of a nick by the replisome of bacteriophage T7

DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick.     

The functional role of a DNA primase in chloroplast DNA replication in Chlamydomonas reinhardtii.

A complementation experiment was developed to identify the protein component that is essential for the in vitro replication of a cloned template containing a chloroplast DNA replication origin of Chlamydomonas reinhardtii. Using this method, we have identified a DNA primase activity that copurified with DNA polymerase from the crude protein mixture. The primase catalyzed the synthesis of short RNA primers on single-stranded DNA templates. Among the synthetic templates, the order of preference was poly(dA), poly(dT), and poly(dC). The primer size range for these templates was 11-18, 5-12, and 3-11 nucleotides, respectively. On a single-stranded template containing the chloroplast DNA replication origin, the primer length range reached 19 to 27 nucleotides, indicating a better processtivity. Several initiation sites were mapped on both strands of the cloned replication origin. Some preferential initiation sites were located on A tracks spaced at one helical turn apart within the bending locus. Primase improved the template specificity of the in vitro DNA replication system and enhanced the incorporation of radioactive dATP into the supercoiled template containing the core sequences of the chloroplast DNA replication origin.     

Interrogation of multimeric DNA amplification products by competitive primer extension using bst DNA polymerase (large fragment).

Linear dsDNA composed of tandem repeats may be exponentially amplified by the strongly strand-displacing Bst DNA polymerase (large fragment) and two primers specific for opposite strands. When the repetitive DNA is derivedfrom rolling circle replication of a circular template, the reaction is termed cascade rolling circle amplification (CRCA). We have developed a variant of CRCA in which one primer is attached to the surface of a microwell and the other is labeled, thus enabling detection of amplified material using an ELISA-like protocol. The circular template is derived by annealing and ligation of a padlock on target DNA. It was found that there was good correlation between the synthesis of amplified material and signal. The specificity of the reaction with respect to single-nucleotide polymorphisms was investigated, and it was found that Bst DNA polymerase is prone to extension from primers with mismatched 3' ends. Reliable single nucleotide specificity was only obtained when pre-synthesized amplified material was interrogated by competitive primer extension.     

A role for single-stranded templates in cell-free adeno-associated virus DNA replication

Assays have been described in which duplex adeno-associated virus (AAV) DNA can be replicated in HeLa cell extracts with exogenous AAV Rep protein. These assays appear to mimic the AAV DNA replication that occurs in the cell, including the ability of extracts from adenovirus (Ad)-infected cells to replicate duplex AAV DNA templates more efficiently than extracts from uninfected cells can. We showed previously that the Ad-infected extract was able to support a more processive replication than the uninfected extract. When the Ad single-stranded DNA binding protein (Ad-DBP) was added to an uninfected extract, DNA replication became processive. Based on a strand displacement replication model, we hypothesized that the Ad-DBP was stabilizing the displaced single-stranded DNA during strand displacement replication. In this report, we show that in Ad-infected extracts most of the newly replicated duplex DNA is converted into a single-stranded form shortly after synthesis. Using the results of assays for the replication of single-stranded AAV DNA, we show that these single-stranded molecules serve as templates for additional replication. In addition, we identify a class of molecules which are likely to be intermediates of replication on single-stranded templates. We discuss a possible role for replication of single-stranded molecules in the infected cell.     

Bacteriophage phi X174 RF DNA replication in vivo. A study by electron microscopy.

We have investigated bacteriophage φX174 RF ² DNA replication by electron microscopy. Three different, types of replicative intermediates were observed: rolling circles, partially duplex DNA circles and structures consisting of two DNA circles connected at a single point.Rolling circles with a single-stranded or partially double-stranded DNA tail were both observed. After cleavage of the rolling circles with the restriction endonuclease from Providentia stuartii 164 (PstI) the startpoint of rolling circle replication could be located at 21 map units from the PstI cleavage site in agreement with the previously determined position of the origin of φX RF DNA replication.Partially duplex DNA circles consist of circular viral DNA strands and incomplete complementary DNA strands. After cleavage of these molecules with PstI information about the startpoints of the synthesis of the complementary DNA strand was obtained.The connected DNA circles always contain one completely double-stranded DNA circle whereas the other circle consists of either single-stranded, partially duplex or completely duplex DNA.Part of the duplex-to-duplex DNA circles represent the well-known figure eight or catenated circular dimers. The other connected DNA circles presumably represent replication intermediates which arise by the association of the end of the genome length tail of the rolling circle with the origin-terminus region. This is suggested by the fact that the point of contact between the two DNA circles is located at approximately 21 map units from the Pst1 cleavage site, i.e. at the origin-terminus region of the φX genome. The connected DNA circles may be intermediates in the circularization and cleavage of the genome-length tail of the rolling circles in vivo.A model for φX174 RF DNA replication in vivo summarizing the data obtained by biochemical (Baas et al., 1978) and electron microscopic analysis of replicative intermediates is presented (Fig. 9).     

Enzymatic Mechanisms of DNA Replication

DNA polymerases purified from several sources are characterized by replication of the 3'-hydroxy-terminated strand of a helical template. Failure to achieve simultaneous replication of the 5'-strand leads to aberrations in the synthesized DNA, described as nondenaturability and branching. Aberrations in synthesized DNA were not observed when (a) the 5'-strand was destroyed by a specific nuclease during the course of replication or (b) a single-stranded (circular) phage (M13) DNA served as template. Replication of a single-stranded, circular DNA produced a helical product, but the nature of initiation of a new strand by the circular template remains to be explained. Hypothetical mechanisms for simultaneous replication of the 5'-strand are presented as is the possibility that the tertiary structure of the DNA, as for example, a circular form of the helix, is of prime importance in in vivo replication.     

In vitro replication of bacteriophage PRD1 DNA. Characterization of the protein-primed initiation site.

Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Using single-stranded oligonucleotide templates carrying the sequence corresponding to the 25 first bases of the 3' end of PRD1 DNA, and Mg2+ as the activating metal ion of the phage DNA polymerase, we show that the fourth base from the 3' end of the template directs, by base complementarity, the dNMP to be linked to the phage terminal protein (TP) in the initiation reaction. This result suggests that phage PRD1 maintains its 3' end DNA sequences via a sliding-back mechanism. The single-stranded DNA templates could not be replicated by the PRD1 DNA polymerase, much in contrast to the natural TP-DNA. Nevertheless, the analysis of the transition products obtained with TP-DNA and origin-containing oligonucleotides suggests that sliding-back occurs stepwise, the fourth base being the directing position during the entire process.     

Discontinuous DNA synthesis by purified mammalian proteins

Five proteins purified from mouse cells acting together efficiently convert a single-stranded circular DNA template to covalently closed duplex circle by a discontinuous mechanism. DNA polymerase alpha/primase with the assistance of alpha accessory factor covers the single-stranded circle with RNA-primed DNA fragments. Primers are removed by a combination of RNase H-1 and a 5'-exonuclease that was identified by its ability to complete this in vitro system. The 5'-exonuclease is required to remove residual one or two ribonucleotides at the primer/DNA junction that are resistant to RNase H-1. Gap filling is by the DNA polymerase alpha/primase, and DNA ligase I converts the DNA fragments to continuous strand. The concerted action of the five proteins emulates synthesis of the staging strand at the replication fork.     

Rolling circle amplification of genomic templates for inverse PCR (RCA–GIP): a method for 5′- and 3′-genome walking without anchoring

We have devised an improved method of genome walking, named rolling circle amplification of genomic templates for Inverse PCR (RCA–GIP). The method is based on the generation of circular genomic DNA fragments, followed by rolling circle amplification of the circular genomic DNA using ϕ29 DNA polymerase without need for attachment of anchor sequences. In this way from the circular genomic DNA fragments, after RCA amplification, a large amount of linear concatemers is generated suitable for Inverse PCR template that can be amplified, sequenced or cloned allowing the isolation of the 3′- and 5′- of unknown ends of genomic sequences. To prove the concept of the proposed methodology, we used this procedure to isolate the promoter regions from different species. Herein as an example we present the isolation of four promoter regions from Crocus sativus, a crop cultivated for saffron production.     

The ability of DNA polymerase beta to synthesize DNA beyond the gap with displacement of the non-replicated strand

Study was made of the ability of calf thymus DNA polymerases alpha and beta to replicate templates containing a small gap. It was found that during extensive replication of activated DNA or synthetic template.primers or specially prepared circular DNA containing a small gap, catalyzed by DNA polymerase alpha, the levels of incorporated nucleotides corresponded to the amounts of the single-stranded fraction of these templates. In contrast, in the reaction catalyzed by DNA polymerase beta the amounts of products were several times greater. The ability to synthesize the product in a great excess was a specific feature of the latter enzyme. An analysis of the gap-filling products by sucrose gradient centrifugation, gel electrophoresis and Southern hybridization showed that, contrary to DNA polymerase alpha, DNA polymerase beta exhibited the ability to synthesize DNA not only within but also beyond the gap. The "net" DNA product is complementary to the template strand. It is suggested that DNA was synthesized beyond the gap by displacement of the non-replicated strand.     

Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits

DNA polymerase III of Escherichia coli requires multiple auxiliary factors to enable it to serve as a replicative complex. We demonstrate that auxiliary components of the DNA polymerase III holoenzyme, the gamma delta complex and beta subunit, markedly stimulate DNA polymerase II on long single-stranded templates. DNA polymerase II activity is enhanced by single-stranded DNA binding protein, but the stimulation by gamma delta and beta can be observed either in the absence or presence of single-stranded DNA binding protein. In contrast with DNA polymerase III, the requirement of DNA polymerase II for gamma delta cannot be bypassed by large excesses of the beta subunit at low ionic strength in the absence of the single-stranded DNA binding protein. The product of the DNA polymerase II-gamma delta-beta reaction on a uniquely primed single-stranded circle is of full template length; the reconstituted enzyme apparently is incapable of strand displacement synthesis. The possible biological implications of these observations are discussed.