DNA polymerase III from Saccharomyces cerevisiae. II. Inhibitor studies and comparison with DNA polymerases I and II

The newly identified yeast DNA polymerase III was compared to DNA polymerases I and II and the mitochondrial DNA polymerase. Inhibition by aphidicolin (I50) of DNA polymerases I, II, and III was 4, 6, and 0.6 micrograms/ml, respectively. The mitochondrial enzyme was insensitive to the drug. N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate strongly inhibited DNA polymerase I (I50 = 0.3 microM), whereas DNA polymerase III was less sensitive (I50 = 80 microM). Conditions that allowed proteolysis to proceed during the preparation of extracts converted DNA polymerase II from a sensitive form (I50 = 2.4 microM) to a resistant form (I50 = 2 mM). The mitochondrial DNA polymerase is insensitive (I50 greater than 5 mM). With most other inhibitors tested (N-ethylmaleimide, heparin, salt) only small differences were observed between the three nuclear DNA polymerases. Polyclonal antibodies to DNA polymerase III did not inhibit DNA polymerases I and II, nor were those polymerases recognized by Western blotting. Monoclonal antibodies to DNA polymerase I did not crossreact with DNA polymerases II and III. The results show that DNA polymerase III is distinct from DNA polymerase I and II.     

DNA polymerase III from Saccharomyces cerevisiae. I. Purification and characterization

Yeast cells from a wild type or protease-deficient strain were lysed in the absence or presence of protease inhibitors and the extracts analyzed by analytical high pressure liquid chromatography on diethylaminoethyl silica gel. Conditions that inhibited protease action caused elution of a novel DNA polymerase activity at a position in the gradient distinct from the elution positions of both DNA polymerase I and II. In large scale purifications, this DNA polymerase, called DNA polymerase III, copurified with a single-stranded DNA dependent 3'-5' exonuclease activity, exonuclease III, to near homogeneity. Glycerol gradient centrifugation partially dissociated the complex to yield two peaks of exonuclease III activity, one at 7.7 S together with the DNA polymerase, and one at 4.0 S without polymerase activity. Gel filtration indicated that the complex has a molecular mass greater than 400 kDa. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the complex consists of several subunits: 140, 62, 55, and 53 kilodaltons, some of which may be proteolysis products. The exonuclease component of the complex can excise single nucleotide mismatches providing a base-paired primer-template which can be elongated by the DNA polymerase. Under replication conditions, the complex exhibits a measurable turnover rate of dTTP to dTMP and it contains no primase activity. The enzymatic activities of the 3'-5' exonuclease are consistent with a proofreading function during in vivo DNA replication. A second exonuclease activity, exonuclease IV, separated from the complex late in the purification scheme. It degrades both single-stranded and double-stranded DNA in the 5'----3' direction.     

FTMS and TOF/TOF mass spectrometry in concert: identifying peptides with high reliability using matrix prespotted MALDI target plates

In this paper we describe a combination of the mass spectrometric techniques MALDI-TOF/TOF and MALDI-FTMS to identify proteins in complex samples using prespotted MALDI target plates. By this procedure accurate FTMS mass measurements and TOF/TOF data are obtained from the same spot. We have found that this combination of techniques leads to more reliable identification of peptides.     

Incorporating a gender perspective into the development of clinical guidelines: a training course for guideline developers

Background

The Research-Based Education and Quality Improvement (ReBEQI) European partnership aims to establish a framework and provide practical tools for the selection, implementation, and evaluation of quality improvement (QI) interventions. We describe the development and preliminary evaluation of the software tool NorthStar, a major product of the ReBEQI project.

Methods

We focused the content of NorthStar on the design and evaluation of QI interventions. A lead individual from the ReBEQI group drafted each section, and at least two other group members reviewed it. The content is based on published literature, as well as material developed by the ReBEQI group. We developed the software in both a Microsoft Windows HTML help system version and a web-based version. In a preliminary evaluation, we surveyed 33 potential users about the acceptability and perceived utility of NorthStar.

Results

NorthStar consists of 18 sections covering the design and evaluation of QI interventions. The major focus of the intervention design sections is on how to identify determinants of practice (factors affecting practice patterns), while the major focus of the intervention evaluation sections is on how to design a cluster randomised trial. The two versions of the software can be transferred by email or CD, and are available for download from the internet. The software offers easy navigation and various functions to access the content. Potential users (55% response rate) reported above-moderate levels of confidence in carrying out QI research related tasks if using NorthStar, particularly when developing a protocol for a cluster randomised trial

Conclusion

NorthStar is an integrated, accessible, practical, and acceptable tool to assist developers and evaluators of QI interventions.     

Human Exonuclease 5 Is a Novel Sliding Exonuclease Required for Genome Stability

Previously, we characterized Saccharomyces cerevisiae exonuclease 5 (EXO5), which is required for mitochondrial genome maintenance. Here, we identify the human homolog (C1orf176; EXO5) that functions in the repair of nuclear DNA damage. Human EXO5 (hEXO5) contains an iron-sulfur cluster. It is a single-stranded DNA (ssDNA)-specific bidirectional exonuclease with a strong preference for 5′-ends. After loading at an ssDNA end, hEXO5 slides extensively along the ssDNA prior to cutting, hence the designation sliding exonuclease. However, the single-stranded binding protein human replication protein A (hRPA) restricts sliding and enforces a unique, species-specific 5′-directionality onto hEXO5. This specificity is lost with a mutant form of hRPA (hRPA-t11) that fails to interact with hEXO5. hEXO5 localizes to nuclear repair foci in response to DNA damage, and its depletion in human cells leads to an increased sensitivity to DNA-damaging agents, in particular interstrand cross-linking-inducing agents. Depletion of hEXO5 also results in an increase in spontaneous and damage-induced chromosome abnormalities including the frequency of triradial chromosomes, suggesting an additional defect in the resolution of stalled DNA replication forks in hEXO5-depleted cells.     

Refined identification of neutralization-resistant HIV-1 CRF02_AG viruses

We studied neutralization of CRF02_AG HIV-1-infected plasma samples. In contrast to previous reports, these samples neutralized CRF02_AG viruses better than other viruses. This included six of eight CRF02_AG viruses previously designated resistant (tier 2/3 or 3). Only viruses 253-11 and 278-50 remained highly resistant, but they were sensitive to membrane-proximal external region (MPER)-specific monoclonal antibodies, suggesting neutralization targets for even these viruses. We propose using high-neutralizing-within-subtype samples for evaluation of neutralization resistance of viruses.     

A Small Novel A-Kinase Anchoring Protein (AKAP) That Localizes Specifically Protein Kinase A-Regulatory Subunit I (PKA-RI) to the Plasma Membrane

Protein kinase A-anchoring proteins (AKAPs) provide spatio-temporal specificity for the omnipotent cAMP-dependent protein kinase (PKA) via high affinity interactions with PKA regulatory subunits (PKA-RI, RII). Many PKA-RII-AKAP complexes are heavily tethered to cellular substructures, whereas PKA-RI-AKAP complexes have remained largely undiscovered. Here, using a cAMP affinity-based chemical proteomics strategy in human heart and platelets, we uncovered a novel, ubiquitously expressed AKAP, termed small membrane (sm)AKAP due to its specific localization at the plasma membrane via potential myristoylation/palmitoylation anchors. In vitro binding studies revealed specificity of smAKAP for PKA-RI (K_d = 7 nm) over PKA-RII (K_d = 53 nm) subunits, co-expression of smAKAP with the four PKA R subunits revealed an even more exclusive specificity of smAKAP for PKA-RIα/β in the cellular context. Applying the singlet oxygen-generating electron microscopy probe miniSOG indicated that smAKAP is tethered to the plasma membrane and is particularly dense at cell-cell junctions and within filopodia. Our preliminary functional characterization of smAKAP provides evidence that, like PKA-RII, PKA-RI can be tightly tethered by a novel repertoire of AKAPs, providing a new perspective on spatio-temporal control of cAMP signaling.     

RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase delta

In fulfilling its biosynthetic roles in nuclear replication and in several types of repair, DNA polymerase δ (pol δ) is assisted by replication protein A (RPA), the single-stranded DNA-binding protein complex, and by the processivity clamp proliferating cell nuclear antigen (PCNA). Here we report the effects of these accessory proteins on the fidelity of DNA synthesis in vitro by yeast pol δ. We show that when RPA and PCNA are included in reactions containing pol δ, rates for single base errors are similar to those generated by pol δ alone, indicating that pol δ itself is by far the prime determinant of fidelity for single base errors. However, the rate of deleting multiple nucleotides between directly repeated sequences is reduced by ~10-fold in the presence of either RPA or PCNA, and by ≥90-fold when both proteins are present. We suggest that PCNA and RPA suppress large deletion errors by preventing the primer terminus at a repeat from fraying and/or from relocating and annealing to a downstream repeat. Strong suppression of deletions by PCNA and RPA suggests that they may contribute to the high replication fidelity needed to stably maintain eukaryotic genomes that contain abundant repetitive sequences.     

Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase ζ

To probe Pol ζ functions in vivo via its error signature, here we report the properties of Saccharomyces cerevisiae Pol ζ in which phenyalanine was substituted for the conserved Leu-979 in the catalytic (Rev3) subunit. We show that purified L979F Pol ζ is 30% as active as wild-type Pol ζ when replicating undamaged DNA. L979F Pol ζ shares with wild-type Pol ζ the ability to perform moderately processive DNA synthesis. When copying undamaged DNA, L979F Pol ζ is error-prone compared to wild-type Pol ζ, providing a biochemical rationale for the observed mutator phenotype of rev3-L979F yeast strains. Errors generated by L979F Pol ζ in vitro include single-base insertions, deletions and substitutions, with the highest error rates involving stable misincorporation of dAMP and dGMP. L979F Pol ζ also generates multiple errors in close proximity to each other. The frequency of these events far exceeds that expected for independent single changes, indicating that the first error increases the probability of additional errors within 10 nucleotides. Thus L979F Pol ζ, and perhaps wild-type Pol ζ, which also generates clustered mutations at a lower but significant rate, performs short patches of processive, error-prone DNA synthesis. This may explain the origin of some multiple clustered mutations observed in vivo.