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Biomedical subjects

M Stucki

Publications and source records attributed to M Stucki.

At least 19 recordsLinked to original sources
PubMed2001

Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase delta by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1.

DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol delta coupled to DNA synthesis. Our results suggested that pol delta alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol delta, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a "locked" flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase delta is modulated by the concerted action of PCNA, RP-A and Fen 1.

Animals↗
PubMed2001

A coordinated interplay: proteins with multiple functions in DNA replication, DNA repair, cell cycle/checkpoint control, and transcription.

In eukaryotic cells, DNA transactions such as replication, repair, and transcription require a large set of proteins. In all of these events, complexes of more than 30 polypetides appear to function in highly organized and structurally well-defined machines. We have learned in the past few years that the three essential macromolecular events, replication, repair, and transcription, have common functional entities and are coordinated by complex regulatory mechanisms. This can be documented for replication and repair, for replication and checkpoint control, and for replication and cell cycle control, as well as for replication and transcription. In this review we cover the three different protein classes: DNA polymerases, DNA polymerase accessory proteins, and selected transcription factors. The "common enzyme-different pathway strategy" is fascinating from several points of view: first, it might guarantee that these events are coordinated; second, it can be viewed from an evolutionary angle; and third, this strategy might provide cells with backup mechanisms for essential physiological tasks.

Cell Cycle↗
PubMed2001

Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300.

We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a complex with flap endonuclease-1 (Fen1) and acetylated Fen1 in vitro. Furthermore, Fen1 acetylation was observed in vivo and was enhanced upon UV treatment of human cells. Remarkably, acetylation of the Fen1 C terminus by p300 significantly reduced Fen1's DNA binding and nuclease activity. Proliferating cell nuclear antigen (PCNA) was able to stimulate both acetylated and unacetylated Fen1 activity to the same extent. Our results identify acetylation as a novel regulatory modification of Fen1 and implicate that p300 is not only a component of the chromatin remodeling machinery but might also play a critical role in regulating DNA metabolic events.

Acetylation↗
PubMed2000

In eukaryotic flap endonuclease 1, the C terminus is essential for substrate binding.

Flap endonuclease 1 (Fen1) is a structure-specific metallonuclease with important functions in DNA replication and DNA repair. It interacts like many other proteins involved in DNA metabolic events with proliferating cell nuclear antigen (PCNA), and its enzymatic activity is stimulated by PCNA in vitro. The PCNA interaction site is located close to the C terminus of Fen1 and is flanked by a conserved basic region of 35-38 amino acids in eukaryotic species but not in archaea. We have constructed two deletion mutants of human Fen1 that lack either the PCNA interaction motif or a part of its adjacent C-terminal region and analyzed them in a variety of assays. Remarkably, deletion of the basic C-terminal region did not affect PCNA interaction but resulted in a protein with significantly reduced enzymatic activity. Electrophoretic mobility shift analysis revealed that this mutant displayed a severe defect in substrate binding. Our results suggest that the C terminus of eukaryotic Fen1 consists of two functionally distinct regions that together might form an important regulatory domain.

Amino Acid Sequence↗
PubMed2000

Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK.

During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues are replaced by repair synthesis. A set of proteins required for repair of most lesions is RPA, XPA, TFIIH, XPC-hHR23B, XPG, and ERCC1-XPF, but additional components have not been excluded. The most complex and difficult to analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in basal transcription initiation and in DNA repair, and several inherited human disorders are associated with mutations in TFIIH subunits. To identify the forms of TFIIH that can function in repair, recombinant XPA, RPA, XPC-hHR23B, XPG, and ERCC1-XPF were combined with TFIIH fractions purified from HeLa cells. Repair activity coeluted with the peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was active. Recombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indicating that CAK can negatively regulate NER by phosphorylation. The 15 recombinant polypeptides define the minimal set of proteins required for dual incision of DNA containing a cisplatin adduct. Complete repair was achieved by including highly purified human DNA polymerase delta or epsilon, PCNA, RFC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication proteins.

Cyclin-Dependent Kinases↗
PubMed2000

DNA polymerase switching: I. Replication factor C displaces DNA polymerase alpha prior to PCNA loading.

An important not yet fully understood event in DNA replication is the DNA polymerase (pol) switch from pol alpha to pol delta. Indirect evidence suggested that the clamp loader replication factor C (RF-C) plays an important role, since a replication competent protein complex containing pol alpha, pol delta and RF-C could perform pol switching in the presence of proliferating cell nuclear antigen (PCNA). By using purified pol alpha/primase, pol delta, RF-C, PCNA and RP-A we show that: (i) RF-C can inhibit pol alpha in the presence of ATP prior to PCNA loading, (ii) RF-C decreases the affinity of pol alpha for the 3'OH primer ends, (iii) the inhibition of pol alpha by RF-C is released upon PCNA loading, (iv) ATP hydrolysis is required for PCNA loading and subsequent release of inhibition of pol alpha, (v) under these conditions a switching from pol alpha/primase to pol delta is evident. Thus, RF-C appears to be critical for the pol alpha to pol delta switching. Based on these results, a model is proposed in which RF-C induces the pol switching by sequestering the 3'-OH end from pol alpha and subsequently recruiting PCNA to DNA.

Adenosine Triphosphate↗
PubMed2000

The N-terminal region of DNA polymerase delta catalytic subunit is necessary for holoenzyme function.

Genetic and biochemical studies have shown that DNA polymerase delta (Poldelta) is the major replicative Pol in the eukaryotic cell. Its functional form is the holoenzyme composed of Poldelta, proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). In this paper, we describe an N-terminal truncated form of DNA polymerase delta (DeltaN Poldelta) from calf thymus. The DeltaN Poldelta was stimulated as the full-length Poldelta by PCNA in a RF-C-independent Poldelta assay. However, when tested for holoenzyme function in a RF-C-dependent Poldelta assay in the presence of RF-C, ATP and replication protein A (RP-A), the DeltaN Poldelta behaved differently. First, the DeltaN Poldelta lacked holoenzyme functions to a great extent. Second, product size analysis and kinetic experiments showed that the holoenzyme containing DeltaN Poldelta was much less efficient and synthesized DNA at a much slower rate than the holoenzyme containing full-length Poldelta. The present study provides the first evidence that the N-terminal part of the large subunit of Poldelta is involved in holo-enzyme function.

Animals↗
PubMed1999

Long patch base excision repair with purified human proteins. DNA ligase I as patch size mediator for DNA polymerases delta and epsilon.

Among the different base excision repair pathways known, the long patch base excision repair of apurinic/apyrimidinic sites is an important mechanism that requires proliferating cell nuclear antigen. We have reconstituted this pathway using purified human proteins. Our data indicated that efficient repair is dependent on six components including AP endonuclease, replication factor C, proliferating cell nuclear antigen, DNA polymerases delta or epsilon, flap endonuclease 1, and DNA ligase I. Fine mapping of the nucleotide replacement events showed that repair patches extended up to a maximum of 10 nucleotides 3' to the lesion. However, almost 70% of the repair synthesis was confined to 2-4-nucleotide patches and DNA ligase I appeared to be responsible for limiting the repair patch length. Moreover, both proliferating cell nuclear antigen and flap endonuclease 1 are required for the production and ligation of long patch repair intermediates suggesting an important role of this complex in both excision and resynthesis steps.

Base Sequence↗
PubMed1999

Dual mode of interaction of DNA polymerase epsilon with proliferating cell nuclear antigen in primer binding and DNA synthesis.

Proliferating cell nuclear antigen can interact with DNA polymerase epsilon on linear DNA templates, even in the absence of other auxiliary factors (replication factor C, replication protein A), and thereby stimulate its primer recognition and DNA synthesis. Using four characterized mutants of proliferating cell nuclear antigen containing three or four alanine residue substitutions on the C-terminal side and the back side of the trimer, we have tested the kinetics of primer binding and nucleotide incorporation by DNA polymerase epsilon in different assays. In contrast with what has been found in interaction studies between DNA polymerase delta and proliferating cell nuclear antigen, our data suggested that stimulation of DNA polymerase epsilon primer binding involves interactions with both the C-terminal side and the back side of proliferating cell nuclear antigen. However, for stimulation of DNA polymerase epsilon DNA synthesis, exclusively the C-terminal side appears to be sufficient. The significance of this dual interaction is discussed with reference to the physiological roles of DNA polymerase epsilon and its interaction with the clamp proliferating cell nuclear antigen.

Amino Acid Sequence↗
PubMed1998

4'-Acylated thymidine 5'-triphosphates: a tool to increase selectivity towards HIV-1 reverse transcriptase.

4'-Acylated thymidines represent a new class of DNA chain terminators, since they have been shown to act as post-incorporation chain-terminating nucleotides despite the presence of a free 3'-hydroxyl group. Here, we describe the action of the 4'-acetyl- (MeTTP) and 4'-propanoylthymidine 5'-triphosphate (EtTTP) on HIV-1 reverse transcriptase in RNA- and DNA-dependent DNA synthesis and on DNA synthesis catalyzed by the cellular DNA polymerases alpha, beta, delta and epsilon. MeTTP exhibits a high selectivity towards HIV-1 reverse transcriptase. By the use of the bulkier propanoyl group as the 4'-substituent of the nucleoside 5'-triphosphate, selectivity towards HIV-1 reverse transcriptase could be increased without affecting substrate efficiency. Thus, 4'-modifications may serve as a tool to increase selectivity towards HIV-1 reverse transcriptase.

Acylation↗
PubMed1998

Mammalian base excision repair by DNA polymerases delta and epsilon.

Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol beta-deleted mouse fibroblasts to separate subfractions containing either Pol delta or Pol epsilon. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol delta and Pol epsilon were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol beta. Furthermore, our observations indicated, that either Pol delta and/or Pol epsilon participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol delta and/or Pol epsilon are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol beta in one-gap filling reactions.

Animals↗
PubMed1998

Anti-D immunoglobulin in Rh(D) negative volunteers: clearance of Rh(D) positive red cells and kinetics of serum anti-D levels.

Properties of a new anti-D immunoglobulin were assessed in Rh(D) negative healthy male adults. Six volunteers received intravenous, and five volunteers intramuscular injections of 200 micrograms anti-D, 48 hours after pre-treatment with 5 mL of Rh(D) positive erythrocytes. Immediately after intravenous administration of anti-D, a rapid decrease of the Rh(D) positive erythroyctes was noted. After intramuscular injection of anti-D, there was a lag phase of 6 hours until the erythrocytes decreased, and the elimination rate was slower. Twenty-four hours after injection of anti-D, the Rh(D) positive erythrocytes were at the detection limit or no longer detectable in all volunteers. After intravenous administration, anti-D serum levels decreased from 45 ng/mL at 2 hours to 29 ng/mL at 24 hours, whereas after intramuscular administration, anti-D became detectable at 4 hours and increased to 11 ng/mL at 24 hours. During subsequent months, anti-D serum levels decreased at similar rates in both groups. After six months, anti-D was not detectable in any of the volunteers. Thus, the new anti-D immunoglobulin induced elimination of the Rh(D) positive erythrocytes and suggested that Rh(D) immunization of the volunteers was prevented.

Adult↗
PubMed1997

Characterisation of a chromatographically produced anti-D immunoglobulin product.

A chromatographic fractionation method has been developed for the production of a liquid-stable anti-D immunoglobulin product for intravenous and intramuscular use. An immunoglobulin fraction, highly enriched with anti-D immunoglobulins, was isolated by cation-exchange column chromatography and further polished, first by anion-exchange chromatography, followed by an aluminium hydroxide gel treatment. The process includes two specific steps for virus inactivation and removal, namely S/D treatment and nanofiltration. The overall anti-D process yield is about 56%. The final product is stabilised with human albumin and glycine and placed in ready-to-use syringes. The anti-D product was shown to be stable in liquid state for at least 30 months at 4 degrees C.

Chemical Fractionation↗
PubMed1997

The first twelve cases of computer assisted periacetabular osteotomy.

Image guided freehand navigation of surgical instruments has been applied to the Bernese periacetabular osteotomy, a complex surgical technique for the treatment of dysplastic hips. This navigation system has been introduced into the operating room and has so far been used for 12 patients. Image data from computed tomography (CT) scans are presented in various ways to support the preoperative plan and to provide optimized control of surgical action. Special attention has been paid to the implementation of a sophisticated surgeon-machine interface. This paper describes the features of this novel surgical navigation system and its introduction into the clinical environment.

Acetabulum↗
PubMed1996

Basophil histamine release and leukotriene production in response to anti-IgE and anti-IgE receptor antibodies. Comparison of normal subjects and patients with urticaria, atopic dermatitis or bronchial asthma.

The IgE receptor-dependent in vitro mediator release in basophils is characterized by a large interindividual variability both in normal and atopic subjects. The mechanism and the clinical impact of this finding, however, is largely unclear. The aim of the present study was to examine the role of surface-bound IgE and of response-modifying cytokines such as interleukin 3 (IL-3) as possible factors determining basophil releasability in atopic patients and normal controls. Cells from 30 individuals (6 with urticaria, 7 with asthma, 7 with atopic dermatitis, and 30 healthy controls) were isolated and stimulated for mediator release by IL-3 and different triggering antibodies directed against IgE or IgE receptor. Our data suggest that serum IgE levels and basophil receptor occupancy with IgE are not involved in the mechanism of basophil releasability. Furthermore, IL-3-induced similar effects on mediator release in almost all individuals, rather excluding the possibility that releasability is regulated by cytokine priming of basophils. Interestingly, we found that patients with atopic disease have a reduced capacity of releasing mediators upon activation, the mechanism of which is unclear. In conclusion, our findings support the hypothesis that basophil releasability is dependent on cell-imminent mechanisms in basophils, which may be altered in selected atopic patients.

Adult↗
PubMed1989

Production of human antibodies to bee venom phospholipase A2 in vitro.

Phospholipase A2 (PLA) is the major antigen of bee venom. Whereas individuals frequently stung by bees, such as bee keepers, show high levels of IgG4 anti-PLA antibodies in serum, most patients sensitive to bee venom possess increased IgE anti-PLA. We have established a culture system by which anti-PLA antibodies can be induced in vitro. Peripheral blood mononuclear cells were stimulated in a first step with PLA and/or pokeweed mitogen (PWM). After 3 days of culture the cells were washed and further incubated with fresh medium. Anti-PLA antibodies were estimated by an enzyme-linked immunosorbent assay (ELISA) and anti-PLA antibody secreting cells were counted by means of an ELISA plaque assay. Cells from bee keepers, but not those from normal donors, produced anti-PLA IgG in vitro. The isotype pattern of anti-PLA antibodies produced in vitro was identical to that found in serum of the respective donor. Anti-PLA IgM antibody-secreting cells developed at low frequencies in both bee keepers and normal donors whereas PLA-specific IgE could not be detected. The formation of anti-PLA IgG was suppressed by high amounts of antigen, while the IgM response was not affected. We conclude that PWM-induced antibodies reflect the in vivo situation of the donors and that anti-PLA IgG and IgM are regulated by different pathways and mechanisms.

Bee Venoms↗
PubMed1987

Infants' recognition of a face revealed through motion: contribution of internal facial movement and head movement.

The experiment reported here explores 3-month-old infants' ability to recognize a human face from a specific motion pattern lacking static facial features. A woman's face was covered with black makeup and numerous white triangles. It was videotaped while the woman was pretending to interact with a baby. A soft rubber mask was prepared likewise and was videotaped while being moved and deformed by hand. In one condition, the face or mask showed facial movement only, while in a second condition there was internal movement plus head movement. The two stimuli were presented either in upright or in upside-down orientation. Results of 48 subjects indicate that the discrimination of face and mask was easier when the stimuli were presented upright. The absence of head movements did not influence the discriminability. These results suggest that 3-month-old infants organize the moving triangles on the face in the upright orientation into a coherent facelike structure.

Discrimination Learning↗