An alternative approach in gene synthesis: use of long selfpriming oligodeoxynucleotides for the construction of double-stranded DNA

A novel approach for the synthesis of double-stranded DNA fragments from only one long oligodeoxynucleotide (oligo) is presented. The basic strategy is to use oligos which possess a short inverted repeat at their 3' end resulting in the formation of a hairpin structure. The 3' end of this hairpin then serves as a primer in the Klenow (large) fragment of E. coli DNA polymerase I-mediated synthesis of the second DNA strand. Removal of the loop structure as well as generation of sticky ends for subsequent cloning is achieved by digestion with restriction enzymes. Several oligos ranging in size from 130 to 147 nt were synthesized and successfully used in the cloning of gene fragments of up to 120 bp in length. Furthermore, a strategy for the simultaneous cloning of two synthetic DNA fragments is outlined yielding even larger gene fragments. By sequential cloning of these gene fragments the methodology presented here will allow the synthesis of genes of any size. The proposed methodology should also be useful for site-directed mutagenesis as well as saturation mutagenesis.     

Wheat DNA Primase (RNA Primer Synthesis in Vitro,Structural Studies by Photochemical Cross-Linking,and Modulation of Primase Activity by DNA Polymerases)

DNA primase synthesizes short RNA primers used by DNA polymerases to initiate DNA synthesis. Two proteins of approximately 60 and 50 kD were recognized by specific antibodies raised against yeast primase subunits, suggesting a high degree of analogy between wheat and yeast primase subunits. Gel-filtration chromatography of wheat primase showed two active forms of 60 and 110 to 120 kD. Ultraviolet-induced cross-linking with radioactive oligothymidilate revealed a highly labeled protein of 60 kD. After limited trypsin digestion of wheat (Triticum aestivum L.) primase, a major band of 48 kD and two minor bands of 38 and 17 kD were observed. In the absence of DNA polymerases, the purified primase synthesizes long RNA products. The size of the RNA product synthesized by wheat primase is considerably reduced by the presence of DNA polymerases, suggesting a modulatory effect of the association between these two enzymes. Lowering the primase concentration in the assay also favored short RNA primer synthesis. Several properties of the wheat DNA primase using oligoadenylate [oligo(rA)]-primed or unprimed polythymidilate templates were studied. The ability of wheat primase, without DNA polymerases, to elongate an oligo(rA) primer to long RNA products depends on the primer size, temperature, and the divalent cation concentration. Thus, Mn2+ ions led to long RNA products in a very wide range of concentrations, whereas with Mg2+ long products were observed around 15 mM. We studied the ability of purified wheat DNA polymerases to initiate DNA synthesis from an RNA primer: wheat DNA polymerase A showed the highest activity, followed by DNA polymerases B and CII, whereas DNA polymerase CI was unable to initiate DNA synthesis from an RNA primer. Results are discussed in terms of understanding the role of these polymerases in DNA replication in plants.     

Archaeal primase: bridging the gap between RNA and DNA polymerases

In the evolution of life, DNA replication is a fundamental process, by which species transfer their genetic information to their offspring. DNA polymerases, including bacterial and eukaryotic replicases, are incapable of de novo DNA synthesis. DNA primases are required for this function, which is sine qua non to DNA replication. In Escherichia coli, the DNA primase (DnaG) exists as a monomer and synthesizes a short RNA primer. In Eukarya, however, the primase activity resides within the DNA polymerase alpha-primase complex (Pol alpha-pri) on the p48 subunit, which synthesizes the short RNA segment of a hybrid RNA-DNA primer. To date, very little information is available regarding the priming of DNA replication in organisms in Archaea. Available sequenced genomes indicate that the archaeal DNA primase is a homolog of the eukaryotic p48 subunit. Here, we report investigations of a p48-like DNA primase from Pyrococcus furiosus, a hyperthermophilic euryarchaeote. P. furiosus p48-like protein (Pfup41), unlike hitherto-reported primases, does not catalyze by itself the synthesis of short RNA primers but preferentially utilizes deoxynucleotides to synthesize DNA fragments up to several kilobases in length. Pfup41 is the first DNA polymerase that does not require primers for the synthesis of long DNA strands.     

Eukaryotic DNA replication. Enzymes and proteins acting at the fork

A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. model systems for replication, such as the simian virus 40 DNA, lead the way. Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double-helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase delta and the lagging strand discontinuously in small pieces by DNA polymerase alpha. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3'----5'-exonuclease, mostly part of the DNA polymerase delta polypeptide, can perform proof-reading by excising incorrectly base-paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5'----3'-exonuclease, removing the RNA primers, DNA polymerase alpha or beta, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher-order structures like chromatin.     

An in vitro screening technique for DNA polymerases that can incorporate modified nucleotides. Pseudo-thymidine as a substrate for thermostable polymerases.

DNA polymerases are desired that incorporate modified nucleotides into DNA with diminished pausing, premature termination and infidelity. Reported here is a simple in vitro assay to screen for DNA polymerases that accept modified nucleotides based on a set of primer extension reactions. In combination with the scintillation proximity assay (SPA[trade]), this allows rapid and simple screening of enzymes for their ability to elongate oligonucleotides in the presence of unnatural nucleotides. A proof of the concept is obtained using pseudo-thymidine (psiT), the C-nucleoside analog of thymidine, as the unnatural substrate. The conformational properties of psiT arising from the carbon-carbon bond between the sugar and the base make it an interesting probe for the importance of conformational restraints in the active site of polymerases during primer elongation. From a pool of commercially available thermostable polymerases, the assay identified Taq DNA polymerase as the most suitable enzyme for the PCR amplification of oligonucleotides containing psiT. Subsequent experiments analyzing PCR performance and fidelity of Taq DNA polymerase acting on psiT are presented. This is the first time that PCR has been performed with a C-nucleoside.     

Reconstituted Okazaki fragment processing indicates two pathways of primer removal

Eukaryotic Okazaki fragments are initiated by an RNA/DNA primer and extended by DNA polymerase delta (pol delta) and the replication clamp proliferating cell nuclear antigen (PCNA). Joining of the fragments by DNA ligase I to generate the continuous double-stranded DNA requires complete removal of the RNA/DNA primer. Pol delta extends the upstream Okazaki fragment and displaces the downstream RNA/DNA primer into a flap removed by nuclease cleavage. One proposed pathway for flap removal involves pol delta displacement of long flaps, coating of those flaps by replication protein A (RPA), and sequential cleavage of the flap by Dna2 nuclease followed by flap endonuclease 1 (FEN1). A second pathway involves reiterative single nucleotide or short oligonucleotide displacement by pol delta and cleavage by FEN1. We measured the length of FEN1 cleavage products on flaps strand-displaced by pol delta in an oligonucleotide system reconstituted with Saccharomyces cerevisiae proteins. Results showed that in the presence of PCNA and FEN1, pol delta displacement synthesis favors formation and cleavage of primarily short flaps, up to eight nucleotides in length; still, a portion of flaps grows to 20-30 nucleotides. The proportion of long flaps can be altered by mutations in the relevant proteins, sequence changes in the DNA, and reaction conditions. These results suggest that FEN1 is sufficient to remove a majority of Okazaki fragment primers. However, some flaps become long and require the two-nuclease pathway. It appears that both pathways, operating in parallel, are required for processing of all flaps.     

Insight into RNA–DNA primer length counting by human primosome

The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), synthesizes chimeric RNA–DNA primers of a limited length for DNA polymerases delta and epsilon to initiate DNA replication on both chromosome strands. Despite recent structural insights into the action of its two catalytic centers, the mechanism of DNA synthesis termination is still unclear. Here we report results of functional and structural studies revealing how the human primosome counts RNA–DNA primer length and timely terminates DNA elongation. Using a single-turnover primer extension assay, we defined two factors that determine a mature primer length (∼35-mer): (i) a tight interaction of the C-terminal domain of the DNA primase large subunit (p58_C) with the primer 5′-end, and (ii) flexible tethering of p58_C and the DNA polymerase alpha catalytic core domain (p180_core) to the primosome platform domain by extended linkers. The obtained data allow us to conclude that p58_C is a key regulator of all steps of RNA–DNA primer synthesis. The above-described findings provide a notable insight into the mechanism of DNA synthesis termination by a eukaryotic primosome, an important process for ensuring successful primer handover to replication DNA polymerases and for maintaining genome integrity.     

Enzymatic incorporation in DNA of 1,5-anhydrohexitol nucleotides

The ability of several DNA polymerases to catalyze the template-directed synthesis of duplex oligonucleotides containing a base pair between a nucleotide with anhydrohexitol ring and its natural complement has been investigated. All DNA polymerases were able to accept the chemically synthesized anhydrohexitol triphosphate as substrate and to catalyze the incorporation of one anhydrohexitol nucleotide. However, only family B DNA polymerases succeeded in elongating the primer after the incorporation of an anhydrohexitol nucleotide. In this family, Vent (exo(-)) DNA polymerase is the most successful one and was therefore selected for further investigation. Results revealed that at high enzyme concentrations six hATPs could be incorporated; however, a selective incorporation proved only feasible under experimental conditions where no more than two analogues could be inserted. Also the synthesis of a mixed HNA-DNA sequence was examined. Kinetic parameters for incorporation of one anhydrohexitol adenine nucleoside were similar to those of its natural analogue.     

DNA binding properties of the adenovirus DNA replication priming protein pTP

The precursor terminal protein pTP is the primer for the initiation of adenovirus (Ad) DNA replication and forms a heterodimer with Ad DNA polymerase (pol). Pol can couple dCTP to pTP directed by the fourth nucleotide of the viral genome template strand in the absence of other replication proteins, which suggests that pTP/pol binding destabilizes the origin or stabilizes an unwound state. We analyzed the contribution of pTP to pTP/pol origin binding using various DNA oligonucleotides. We show that two pTP molecules bind cooperatively to short DNA duplexes, while longer DNA fragments are bound by single pTP molecules as well. Cooperative binding to short duplexes is DNA sequence independent and most likely mediated by protein/protein contacts. Furthermore, we observed that pTP binds single-stranded (ss)DNA with a minimal length of approximately 35 nt and that random ssDNA competed 25-fold more efficiently than random duplex DNA for origin binding by pTP. Remarkably, short DNA fragments with two opposing single strands supported monomeric pTP binding. pTP did not stimulate, but inhibited strand displacement by the Ad DNA binding and unwinding protein DBP. These observations suggest a mechanism in which the ssDNA affinity of pTP stabilizes Ad pol on partially unwound origin DNA.     

Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication

DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I’s mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases.     

Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta.

DNA synthesis by two eukaryotic DNA polymerases, alpha and delta, was studied using a single-strand M13 DNA template primed at a unique site. In the presence of low amounts of either DNA polymerase alpha or delta, DNA synthesis was limited and short DNA strands of approximately 100 bases were produced. Addition of replication factors RF-A, PCNA and RF-C, which were previously shown to be required for SV40 DNA replication in vitro, differentially stimulated the activity of both DNA polymerases. RF-A and RF-C independently stimulated DNA polymerase alpha activity 4- to 6-fold, yielding relatively short DNA strands (less than 1 kb) and PCNA had no effect. In contrast, polymerase delta activity was stimulated co-operatively by PCNA, RF-A and RF-C approximately 25- to 30-fold, yielding relatively long DNA strands (up to 4 kb). Neither RF-C nor RF-A appear to correspond to known polymerase stimulatory factors. RF-A was previously shown to be required for initiation of DNA replication at the SV40 origin. Results presented here suggest that it also functions during elongation. The differential effects of these three replication factors on DNA polymerases alpha and delta is consistent with the model that the polymerases function at the replication fork on the lagging and leading strand templates respectively. We further suggest that co-ordinated synthesis of these strands requires dynamic protein-protein interactions between these replication factors and the two DNA polymerases.     

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase.

phi29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3'-5' exonuclease active sites. Using single-turnover conditions, we have demonstrated that phi29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that phi29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.     

The control mechanism for lagging strand polymerase recycling during bacteriophage T4 DNA replication

Given the polarity of DNA duplex, replication by the leading strand polymerase is continuous whereas that by the lagging strand polymerase is discontinuous proceeding through Okazaki fragments. Yet the respective polymerases act processively, implying that the recycling of the lagging strand polymerase is a controlled process. We demonstrate that the rate of the lagging strand polymerase relative to that of fork movement affects Okazaki fragment size and generates ssDNA gaps. We show by using a substrate with limited priming sites that Okazaki fragments can be shifted to shorter lengths by varying the rate of the primase. We find that clamp and clamp loader levels affect both primer utilization and Okazaki fragment size, possibly implicating clamp loading onto the RNA primer in the mechanism of lagging strand polymerase recycling. We formulate a signaling model capable of rationalizing the distribution of Okazaki fragments under various conditions for this and possibly other replisomes.     

Replicative DNA Polymerases

In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3′-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein–protein and protein–DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact.The genomes, from the smallest to the largest, provide an enormous challenge for the replicative DNA polymerases to faithfully copy to give the many generations that follow a comparable condition for life. In this article, we discuss the structural and functional bases by which replicative DNA polymerases are able to efficiently and faithfully build new copies of genomes in eubacteria, archaea, and eukaryotes.     

DNA sequencing using differential extension with nucleotide subsets (DENS).

Here we describe template directed enzymatic synthesis of unique primers, avoiding the chemical synthesis step in primer walking. We have termed this conceptually new technique DENS (differential extension with nucleotide subsets). DENS works by selectively extending a short primer, making it a long one at the intended site only. The procedure starts with a limited initial extension of the primer (at 20-30 degrees C) in the presence of only two out of the four possible dNTPs. The primer is extended by 6-9 bases or longer at the intended priming site, which is deliberately selected, (as is the two-dNTP set), to maximize the extension length. The subsequent termination reaction at 60-65 degrees C then accepts the extended primer at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. DENS allows the use of primers as long as 8mers (degenerate in two positions) which prime much more strongly than modular primers involving 5-7mers and which (unlike the latter) can be used with thermostable polymerases, thus allowing cycle-sequencing with dye-terminators compatible with Taq DNA polymerase, as well as making double-stranded DNA sequencing more robust.     

Comparative study of the efficacy of modifying DNA polymerase and DNA matrix by different photoactive groups at the 3'-end of the DNA primer

The dependence of the modification efficiency of DNA polymerases and DNA template on the nature of photoactivatable group and the length of the linker that joins the group with the heterocyclic base of the primer 3'-terminal nucleotide was studied. The primers that contained the photoreactive groups at their 3'-termini were obtained using the rat DNA polymerase beta or the DNA polymerase from Thermus thermophilus in the presence of one of the dTTP analogues carrying the photoreactive group in position 5 of thymidine residue. After irradiating the reaction mixture with UV light and separating the modification products, the level of covalent binding of the [5'-32P]primer to DNA polymerases and template was determined. The primers containing 4-azido-2,5-difluoro-3-chloropyridyl group were shown to be the most effective in the modification of DNA polymerases.     

Seamless gene engineering using RNA- and DNA-overhang cloning

Here we describe two methods for generating DNA fragments with single-stranded overhangs, like those generated by the activity of many restriction enzymes, by simple methods that do not involve DNA digestion. The methods, RNA-overhang cloning (ROC) and DNA-overhang cloning (DOC), generate polymerase chain reaction (PCR) products composed of double-stranded (ds) DNA flanked by single-stranded (ss) RNA or DNA overhangs. The overhangs can be used to recombine DNA fragments at any sequence location, creating "perfect" chimeric genes composed of DNA fragments that have been joined without the insertion, deletion, or alteration of even a single base pair. The ROC method entails using PCR primers that contain regions of RNA sequence that cannot be copied by certain thermostable DNA polymerases. Using such a chimeric primer in PCR would yield a product with a 5' overhang identical to the sequence of the RNA component of the primer, which can be used for directional ligation of the amplified product to other preselected DNA molecules. This method provides complete control over both the length and sequence of the overhangs, and eliminates the need for restriction enzymes as tools for gene engineering.