Plasmid-based lacZalpha assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase

The preparation of a gapped pUC18 derivative, containing the lacZalpha reporter gene in the single-stranded region, is described. Gapping is achieved by flanking the lacZalpha gene with sites for two related nicking endonucleases, enabling the excision of either the coding or non-coding strand. However, the excised strand remains annealed to the plasmid through non-covalent Watson-Crick base-pairing; its removal, therefore, requires a heat-cool cycle in the presence of an exactly complementary competitor DNA. The gapped plasmids can be used to assess DNA polymerase fidelity using in vitro replication, followed by transformation into Escherichia coli and scoring the blue/white colony ratio. Results found with plasmids are similar to the well established method based on gapped M13, in terms of background ( approximately 0.08% in both cases) and the mutation frequencies observed with a number of DNA polymerases, providing validation for this straightforward and technically uncomplicated approach. Several error prone variants of the archaeal family-B DNA polymerase from Pyrococcus furiosus have been investigated, illuminating the potential of the method

The 3′–5′ proofreading exonuclease of archaeal family-B DNA polymerase hinders the copying of template strand deaminated bases

Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer–template junction. When uracil is specifically bound, the polymerase–DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3′–5′ proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer–template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3′ base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer–template junction, restoring the stalling position to +4. Thus the 3′–5′ proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases

Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed

Genome sequencing of the extinct Eurasian wild aurochs, Bos primigenius, illuminates the phylogeography and evolution of cattle

Background Domestication of the now-extinct wild aurochs, Bos primigenius, gave rise to the two major domestic extant cattle taxa, B. taurus and B. indicus. While previous genetic studies have shed some light on the evolutionary relationships between European aurochs and modern cattle, important questions remain unanswered, including the phylogenetic status of aurochs, whether gene flow from aurochs into early domestic populations occurred, and which genomic regions were subject to selection processes during and after domestication. Here, we address these questions using whole-genome sequencing data generated from an approximately 6,750-year-old British aurochs bone and genome sequence data from 81 additional cattle plus genome-wide single nucleotide polymorphism data from a diverse panel of 1,225 modern animals. Results Phylogenomic analyses place the aurochs as a distinct outgroup to the domestic B. taurus lineage, supporting the predominant Near Eastern origin of European cattle. Conversely, traditional British and Irish breeds share more genetic variants with this aurochs specimen than other European populations, supporting localized gene flow from aurochs into the ancestors of modern British and Irish cattle, perhaps through purposeful restocking by early herders in Britain. Finally, the functions of genes showing evidence for positive selection in B. taurus are enriched for neurobiology, growth, metabolism and immunobiology, suggesting that these biological processes have been important in the domestication of cattle. Conclusions This work provides important new information regarding the origins and functional evolution of modern cattle, revealing that the interface between early European domestic populations and wild aurochs was significantly more complex than previously thought

Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil

Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3′–5′ proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase–DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase

A trimeric DNA polymerase complex increases the native replication processivity

DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. The primary DNA replication polymerase from Sulfolobus solfataricus (SsoDpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of DNA. Analytical gel filtration and electrophoretic mobility shift assays establish that initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. Protein chemical crosslinking experiments show that these are specific polymerase–polymerase interactions and not just separate binding events along DNA. Isothermal titration calorimetry and fluorescence anisotropy experiments corroborate these findings and show a stoichiometry where three polymerases are bound to a single DNA substrate. The trimeric polymerase complex significantly increases both the DNA synthesis rate and the processivity of SsoDpo1. Taken together, these results suggest the presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form

Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya

Family B DNA polymerases from archaea such as Pyrococcus furiosus, which live at temperatures ∼100°C, specifically recognize uracil in DNA templates and stall replication in response to this base. Here it is demonstrated that interaction with uracil is not restricted to hyperthermophilic archaea and that the polymerase from mesophilic Methanosarcina acetivorans shows identical behaviour. The family B DNA polymerases replicate the genomes of archaea, one of the three fundamental domains of life. This publication further shows that the DNA replicating polymerases from the other two domains, bacteria (polymerase III) and eukaryotes (polymerases δ and ε for nuclear DNA and polymerase γ for mitochondrial) are also unable to recognize uracil. Uracil occurs in DNA as a result of deamination of cytosine, either in G:C base-pairs or, more rapidly, in single stranded regions produced, for example, during replication. The resulting G:U mis-pairs/single stranded uracils are promutagenic and, unless repaired, give rise to G:C to A:T transitions in 50% of the progeny. The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil

Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand

Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process