A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated-naphthoylated DEAE-cellulose, resulting in a low background mutation frequency (~1 × 10(-4)). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system

Archaeal DNA Polymerase-B as a DNA Template Guardian: Links between Polymerases and Base/Alternative Excision Repair Enzymes in Handling the Deaminated Bases Uracil and Hypoxanthine

In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil); Endonuclease V (EndoV, which recognises hypoxanthine); and Endonuclease Q (EndoQ), (which recognises both uracil and hypoxanthine). Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primer-templates containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNA mutations

Novel inhibition of archaeal family-D DNA polymerase by uracil.

International audienceArchaeal family-D DNA polymerase is inhibited by the presence of uracil in DNA template strands. When the enzyme encounters uracil, following three parameters change: DNA binding increases roughly 2-fold, the rate of polymerization slows by a factor of ≈ 5 and 3'-5' proof-reading exonuclease activity is stimulated by a factor of ≈ 2. Together these changes result in a significant decrease in polymerization activity and a reduction in net DNA synthesis. Pol D appears to interact with template strand uracil irrespective of its distance ahead of the replication fork. Polymerization does not stop at a defined location relative to uracil, rather a general decrease in DNA synthesis is observed. 'Trans' inhibition, the slowing of Pol D by uracil on a DNA strand not being replicated is also observed. It is proposed that Pol D is able to interact with uracil by looping out the single-stranded template, allowing simultaneous contact of both the base and the primer-template junction to give a polymerase-DNA complex with diminished extension ability

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

Template-directed addition of nucleosides to DNA by the BfiI restriction enzyme

Restriction endonucleases catalyse DNA cleavage at specific sites. The BfiI endonuclease cuts DNA to give staggered ends with 1-nt 3′-extensions. We show here that BfiI can also fill in the staggered ends: while cleaving DNA, it can add a 2′-deoxynucleoside to the reaction product to yield directly a blunt-ended DNA. We propose that nucleoside incorporation proceeds through a two-step reaction, in which BfiI first cleaves the DNA to make a covalent enzyme–DNA intermediate and then resolves it by a nucleophilic attack of the 3′-hydroxyl group of the incoming nucleoside, to yield a transesterification product. We demonstrate that base pairing of the incoming nucleoside with the protruding DNA end serves as a template for the incorporation and governs the yield of the elongated product. The efficiency of the template-directed process has been exploited by using BfiI for the site-specific modification of DNA 5′-termini with an amino group using a 5′-amino-5′-deoxythymidine

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

Alcohol Intake Between Menarche and First Pregnancy: A Prospective Study of Breast Cancer Risk

Background: Adult alcohol consumption during the previous year is related to breast cancer risk. Breast tissue is particularly susceptible to carcinogens between menarche and first full-term pregnancy. No study has characterized the contribution of alcohol consumption during this interval to risks of proliferative benign breast disease (BBD) and breast cancer. Methods: We used data from 91005 parous women in the Nurses’ Health Study II who had no cancer history, completed questions on early alcohol consumption in 1989, and were followed through June 30, 2009, to analyze breast cancer risk. A subset of 60093 women who had no history of BBD or cancer in 1991 and were followed through June 30, 2001, were included in the analysis of proliferative BBD. Relative risks (RRs) were estimated using Cox proportional hazard regression. Results: We identified 1609 breast cancer cases and 970 proliferative BBD cases confirmed by central histology review. Alcohol consumption between menarche and first pregnancy, adjusted for drinking after first pregnancy, was associated with risks of breast cancer (RR = 1.11 per 10g/day intake; 95% confidence interval [CI] = 1.00 to 1.23) and proliferative BBD (RR = 1.16 per 10g/day intake; 95% CI = 1.02 to 1.32). Drinking after first pregnancy had a similar risk for breast cancer (RR = 1.09 per 10g/day intake; 95% CI = 0.96 to 1.23) but not for BBD. The association between drinking before first pregnancy and breast neoplasia appeared to be stronger with longer menarche to first pregnancy intervals. Conclusions: Alcohol consumption before first pregnancy was consistently associated with increased risks of proliferative BBD and breast cancer

Musicians’ perceptions and experiences of using simulation training to develop performance skills

Simulation has been applied as a tool for learning and training in sports, psychology and medicine for some time, but its current use and potential for training musicians is less well understood. The aim of this study was to explore musicians’ perceptions and experiences of using simulated performance environments. Nine conservatory students performed in two simulations, each with interactive virtual elements and vivid environmental cues: a recital with a virtual audience and an audition with virtual judges. Qualitative data were collected through a focus group interview and written reflective commentaries. Thematic analysis highlighted the musicians’ experiences in terms of (1) their anticipation of using the simulations, (2) the process of performing in the simulations, (3) the usefulness of simulation as a tool for developing performance skills and (4) ways of improving simulation training. The results show that while simulation was new to the musicians and individual levels of immersion differed, the musicians saw benefits in the approach for developing, experimenting with and enhancing their performance skills. Specifically, the musicians emphasised the importance of framing the simulation experience with plausible procedures leading to and following on from the performance, and they recognised the potential for combining simulation with complementary training techniques

An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family

A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol zeta (a family-B translesion synthesis polymerase). Inactivation of the 3'–5' proofreading exonuclease activity is also necessary. The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers. Tgo-Pol Z1 is also proficient at extending from primers that terminate opposite aberrant bases. The fidelity of Tgo-Pol Z1 is reduced, with amarked tendency tomake changes at G:C base pairs. Together, these results suggest that the loop region of the fingers domain may play a critical role in determining whether a family-B enzyme falls into the accurate genome-replicating category or is an errorprone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA

Study of the reaction e^{+}e^{-} -->J/psi\pi^{+}\pi^{-} via initial-state radiation at BaBar

We study the process $e^+e^-\to J/\psi\pi^{+}\pi^{-}$ with initial-state-radiation events produced at the PEP-II asymmetric-energy collider. The data were recorded with the BaBar detector at center-of-mass energies 10.58 and 10.54 GeV, and correspond to an integrated luminosity of 454 $\mathrm{fb^{-1}}$. We investigate the $J/\psi \pi^{+}\pi^{-}$ mass distribution in the region from 3.5 to 5.5 $\mathrm{GeV/c^{2}}$. Below 3.7 $\mathrm{GeV/c^{2}}$ the $\psi(2S)$ signal dominates, and above 4 $\mathrm{GeV/c^{2}}$ there is a significant peak due to the Y(4260). A fit to the data in the range 3.74 -- 5.50 $\mathrm{GeV/c^{2}}$ yields a mass value $4244 \pm 5$ (stat) $\pm 4$ (syst)$\mathrm{MeV/c^{2}}$ and a width value $114 ^{+16}_{-15}$ (stat)$\pm 7$(syst)$\mathrm{MeV}$ for this state. We do not confirm the report from the Belle collaboration of a broad structure at 4.01 $\mathrm{GeV/c^{2}}$. In addition, we investigate the $\pi^{+}\pi^{-}$ system which results from Y(4260) decay