Biomedical subjects

R Knippers

Publications and source records attributed to R Knippers.

At least 109 records · Page 6Linked to original sources

PubMed1986

Synthesis of DNA polymerase alpha analyzed by immunoprecipitation from synchronously proliferating cells.

Synchronously proliferating TC7 monkey and 3T3 mouse cells were pulse labeled with [35S]methionine. Radioactively labeled DNA polymerase alpha was immunoprecipitated with polymerase-specific monoclonal antibodies. The precipitated polypeptides were identified by gel electrophoresis and fluorography. The increase in DNA polymerase alpha activity during S phase was accompanied by an increased synthesis of the enzyme. Some DNA polymerase alpha was synthesized in growth-arrested TC7 cells whereas the synthesis of the large polymerase subunit in 3T3 cells was strictly coupled to the replicative phase of the cell cycle. We also found that DNA polymerase alpha was more prone to proteolysis in TC7 cells than in 3T3 cells. In 3T3 cells, a polymerase subunit with an apparent molecular weight of 186 000 was observed; this subunit was most probably associated with two smaller subunits of Mr 74 000 and 52 000. Synthesis of these three polymerase-associated polypeptides appeared to be regulated differently.

Animals↗

PubMed1986

DNA helicase activity of SV40 large tumor antigen.

Large tumor antigen (T antigen) was extracted from SV40-infected African Green Monkey cells and purified to homogeneity by immunoaffinity chromatography. The purified T antigen preparations unwind DNA duplices of greater than 120 bp in a reaction which is dependent on magnesium ions and ATP hydrolysis. Based on these and other properties of the reaction we classify this newly discovered enzymatic activity as a eukaryotic DNA helicase. The helicase and the known ATPase function of T antigen cosediment with the mono- or dimeric 4-6 S form of T antigen, but not with higher T antigen aggregates. The helicase activity seems to be an intrinsic function of SV40 T antigen. First, several different T antigen-specific monoclonal antibodies interfere with the DNA unwinding activity; monoclonals which are known to reduce the T antigen-specific ATPase most strongly inhibited the helicase reaction. Second, mutant T antigens with impaired ATPase function also showed a reduced DNA unwinding activity.

Animals↗

PubMed1985

A newly detected class of mammalian single strand-specific DNA-binding proteins. Effects on DNA polymerase alpha-catalyzed DNA synthesis.

Using a rapid purification scheme, we have isolated from calf thymus cells two single strand-specific DNA-binding proteins, with apparent molecular masses of 48 and 61 kDa. These proteins remained undetected in earlier studies on DNA-binding proteins from calf thymus because they are extremely sensitive to proteolytic breakdown occurring during purification. The proteins are immunologically and biochemically related. They also share a number of functional properties. Both proteins bind noncooperatively to single-stranded DNA and almost totally ignore double-stranded DNA. Both proteins stimulate DNA synthesis catalyzed by mammalian DNA polymerase alpha in the presence of an excess of "activated" DNA as primer-template. The stimulation factor was between 2 and 100 depending on the protein/DNA ratio. We have analyzed this effect in more detail with specifically primed single-stranded phage fd DNA as template and concluded that the proteins block nonproductive polymerase-binding sites on single-stranded DNA sequences, thereby increasing the probability for an association of polymerase with 3'-OH primer ends.

Animals↗

PubMed1985

A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system.

In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin replication.

Animals↗

PubMed1985

Transfection of mouse fibroblast cells with a promoterless herpes simplex virus thymidine kinase gene: number of integrated gene copies and structure of single and amplified gene sequences.

Plasmids carrying the herpes simplex virus thymidine kinase (tk) gene were used to transfect thymidine kinase-deficient cells of the mouse fibroblast cell line LM(tk-). Individual cell clones were cultivated in selective hypoxanthine-aminopterin-thymidine medium to determine the number of integrated plasmid copies which was almost always in the range of one to three copies per genome. In contrast, cells transfected with plasmids carrying a promoterless "truncated" tk gene typically contained between 10 and 25 copies per genome. Surprisingly, when the truncated tk gene was transfected together with a simian virus 40 DNA segment, including its transcriptional enhancer, the number of integrated tk gene copies was always low, between one and three copies per genome. We have analyzed the genomic organization of integrated truncated tk genes by blot hybridization of restricted cellular DNA and concluded that integrated units of plasmid DNA molecules are arranged in tandem arrays which remain stable in most cases for many cell generations. In only 1 of ca. 20 cell clones did we observe a retraction and expansion of the number of integrated promoterless tk genes as a response to the removal or readdition of selective pressure. Surprisingly, the thymidine kinase activity determined in extracts from cells growing in selective hypoxanthine-aminopterin-thymidine medium (high numbers of integrated tk gene copies) was nearly the same as the enzymatic activity in cells growing in nonselective medium (low copy numbers). Moreover, Northern blots of polyadenylated RNA, extracted from cells growing under selective and nonselective conditions, showed that, in both cases, the major species of tk-specific transcripts was ca. 1.5 kilobases in size, as expected for a tk-specific mRNA containing the entire coding region of the gene. Thus, disproportionate DNA replication appeared not to be essential for an active tk gene expression in these cells. We discuss possible pathways leading to the formation of tandem arrays of integrated truncated tk genes and the conditions required for disproportionate DNA replication in the unique case in which we found a retraction and expansion of tk gene copy numbers as a response to selective growth conditions.

Animals↗

PubMed1984

ATP and the processivity of Xenopus laevis DNA polymerase alpha.

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase alpha from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase alpha to DNA and conclude that ATP increases the processivity of the enzyme.

Adenosine Monophosphate↗

PubMed1984

Chromatin structure and DNA replication.

The structure of (3H) thymidine pulse labeled, replicating chromatin differs from that of non replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. We summarize the structural changes that we observe using replicating chromatin as substrate for micrococcal nuclease. The data suggest a more extended configuration of replicating compared to non replicating chromatin. We use these data to discuss a model of the chromatin structure in the vicinity of replication forks. Finally, we present data to show that the reversion of structural changes in replicative chromatin depends on continued DNA replication.

Animals↗

PubMed1984

DNA binding properties of a mutant T antigen from the simian virus 40-transformed human cell line simian virus 80.

We investigated whether the T antigen of the simian virus 40-transformed human cell line simian virus 80 ( SV80 ) specifically recognizes DNA sequences of its own template, i.e, the viral sequences integrated in the SV80 cellular genome. In vitro DNA binding experiments clearly indicated that, in contrast to wild-type T antigen, SV80 T antigen does not specifically bind to sites on the integrated viral DNA in SV80 cells.

Antigens, Polyomavirus Transforming↗

PubMed1983

Reactions in vitro of the DNA polymerase-primase from Xenopus laevis eggs. A role for ATP in chain elongation.

A form of DNA polymerase alpha was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single-stranded phage fd DNA template into a double-stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a greater than 100 000-Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase-primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600-2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase-primase stops at the first stable hairpin structure upstream from the 3'-OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2 mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2-4 times longer DNA strands. Our results suggest a role for ATP in the polymerase-primase-catalyzed chain-elongation reaction.

Adenosine Triphosphate↗

PubMed1983

The simian-virus-40 large-tumor antigen in replicating viral chromatin. A salt-resistant protein-DNA interaction.

The large tumor antigen (T antigen) is a genome regulation protein, coded by simian virus 40, that binds with high affinity to specific binding sites on viral DNA. The specifically bound T antigen is released from these sites in 0.2-0.3 M NaCl. Immunoprecipitation techniques were used to show that T antigen also dissociates in 0.2-0.3 M NaCl from mature viral chromatin but not from replicating viral chromatin. In fact, a considerable fraction of T antigen remains associated with replicating chromatin at NaCl concentrations as high as 1.2 M NaCl when most chromatin proteins, including histones, dissociate. However, T antigen binding to both replicating DNA and mature DNA is sensitive to intercalating drugs such as caffeine and ethidium bromide. We consider the possibility that the unexpectedly tight binding of T antigen to replicating DNA is related to the function that T antigen performs during viral DNA replication.

Antigens, Neoplasm↗

PubMed1983

Chromatin maturation depends on continued DNA-replication.

The structure of [3H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [3H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. We conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation.

Animals↗

PubMed1983

Simian virus 40 large tumor antigen on replicating viral chromatin: tight binding and localization on the viral genome.

Pulse-labeled simian virus 40 (SV40) chromatin as well as uniformly labeled viral chromatin are immunoprecipitable by an SV40-specific tumor antiserum and therefore contain bound tumor antigen (T antigen). Single-stranded calf thymus DNA, immobilized on cellulose, competes effectively for T antigen binding with uniformly labeled nonreplicating, but not with pulse-labeled replicating, chromatin. Furthermore, T antigen dissociates in 0.5 M NaCl from nonreplicating chromatin and from purified SV40 DNA, whereas most T antigen remains associated with replicating chromatin even in the presence of 1.2 to 1.5 M NaCl. We used filtration through DNA-cellulose columns and treatment with high salt to prepare pulse-labeled immunoreactive viral chromatin. The viral DNA was digested before, and in other experiments after, immunoprecipitation with the restriction endonuclease HindIII. We found that SV40 DNA sequences, most probably representing the entire genome, remain in the immunoprecipitate after HindIII digestion, indicating an association of T antigen with origin-distal sections of replicating viral DNA. The results suggest that T antigen in replicating chromatin may be bound to regions close to replicating points. We performed control experiments with in vitro-formed complexes of T antigen and SV40 DNA. When these complexes were immunoprecipitated and HindIII digested we found, in agreement with previous studies, that only the origin containing the HindIII C fragment carried bound T antigen.

Animals↗

PubMed1982

Circular single stranded phage M13-DNA as a template for DNA synthesis in protein extracts from Xenopus laevis eggs: evidence for a eukaryotic DNA priming activity.

Unfractionated protein extracts from activated Xenopus laevis eggs contain all functions required for the chain elongation reactions in replicative DNA synthesis (A.Richter, B. Otto and R.Knippers, 1981, Nucl.Ac.Res. 9, 3793-3807). In order to further explore the DNA synthesizing capacity of this in vitro system and to obtain information on the DNA priming activity in these extracts single stranded phage M13-DNA was used as template for in vitro DNA synthesis. The main results of this investigation are: (i) single stranded circular template DNA is converted to a double stranded DNA form in an alpha-amanitin-insensitive reaction which is absolutely dependent on ribonucleoside triphosphates; (ii) the in vitro synthesized complementary strands are DNA fragments of 1000-2000 nucleotides lengths; (iii) the DNA primase activity copurifies through several column steps and sucrose gradient centrifugation with a DNA polymerase alpha. These activities may therefore be closely associated in a quarternary enzyme complex.

Animals↗

PubMed1982

Subclasses of simian-virus-40 large tumor antigen. Partial purification and DNA-binding properties of two subclasses of tumor antigen from productively infected cells.

Two major subclasses of simian virus 40 tumor antigen were prepared from productively infected monkey cells. These subclasses can be distinguished by their sedimentation properties: one tumor antigen form sediments at 5-6S and the other at 14-16S. The DNA-binding properties of these subclasses were investigated by two different experimental procedures. In the first procedure, the DNA binding of subclasses of crude tumor antigen, separated by zone velocity sedimentation, were assayed by immunoprecipitation of the DNA-protein complexes. In the second procedure, the two tumor antigen forms were partially purified by column chromatography and DNA binding was tested in a filter binding assay. Both procedures gave comparable results. (a) The 5-6-S and the 14-16-S tumor antigen bound specifically to a DNA restriction fragment containing the viral genome control regions. (b) At low salt concentrations, both subclasses bound to specific and to nonspecific DNA sequences; competition experiments in the presence of nonspecific DNA showed, however, that the affinity of both tumor antigen forms for the viral genome control region was at least 10-fold higher than their affinity for nonspecific DNA sequences. (c) The binding of the 5-6-S subclass to viral control region DNA was optimal at 60-80 mM NaCl while specific DNA binding of the 14-16-S form was optimal at 150-200 mM NaCl; however, binding of the 14-16-S form to nonspecific DNA sequences was also more resistant to high salt concentrations than that of the 5-6S form. (d) Both tumor antigen forms bound well to specific and to nonspecific DNA at pH 6-6.5; with increasing pH values, binding to nonspecific DNA decreased while binding to specific DNA reached an optimum at pH 7-7.5. Binding of the 14-16-S form to viral origin DNA was more resistant to pH values above 7.5 than binding of the 5-6-S form.

Animals↗

PubMed1981

A lymphocyte ATP-dependent deoxyribonuclease. Isolation and properties.

An ATP-dependent deoxyribonuclease was isolated from lymphocyte nuclei. The enzyme preparation sediments with about 4 S through sucrose gradients and shows one stainable band after sodium dodecyl sulfate gel electrophoresis. We find three, possibly four, activities associated with the enzyme: a DNA-independent ATPase activity; an ATP-independent endonuclease; an ATP-dependent nuclease which degrades nicked DNA to acid-soluble material; and an unwinding activity producing single-stranded regions in nicked DNA.

Adenosine Triphosphate↗

PubMed1981

A histone H4-specific methyltransferase. Properties, specificity and effects on nucleosomal histones.

A histone H4-specific methyltransferase was purified 80-100-fold from nuclei of calf lymphocytes and from calf thymus. Some biochemical properties of the enzyme are described. The enzyme transfers in vitro methyl groups from S-adenosylmethionine preferentially to the lysine residue 20 of histone H4. This is the major in vivo methylation site of H4. DNA-bound or nucleosomal H4 is not methylated in vitro. We have used methylated and unmodified H4 (in the presence of sufficient quantities of the other core histones) for nucleosome reconstitution in vitro and have not found significant differences in the efficiencies of assembly.

Amino Acids↗

PubMed1981

Replication of SV40 chromatin in extracts from eggs of Xenopus laevis.

Simian virus 40 (SV40) nucleoprotein complexes were prepared from lytically infected cells and used as primer-templates for DNA replication in protein extracts from Xenopus eggs. We found that nucleoprotein containing replicating SV40 DNA served as primer-template while nucleoprotein with nonreplicating SV40 DNA was ineffective. In vitro DNA synthesis begins with short DNA fragments ("Okazaki fragments") which are, in later steps, joined to give unit length SV40 DNA strands, suggesting that in vivo initiated rounds of replication are completed in vitro in the Xenopus system. This conclusion is supported by a restriction enzyme analysis showing that in vitro DNA synthesis occurs in fragments distal to the SV40 origin of replication. Our studies indicate that SV40 DNA replication in Xenopus extracts can be used an an experimental system to study the biochemistry of replicative DNA chain elongation in vitro.

Animals↗

PubMed1981

Asymmetric repair of bacteriophage T7 heteroduplex DNA.

Heteroduplex DNA molecules were prepared in vitro using one strand of DNA carrying a point mutation and one strand of the corresponding wild-type DNA. The heteroduplex DNA was transfected into competent bacteria and the progeny genotypes in the resulting infective centers were determined. From the results we conclude that about 80% of all transfected DNA molecules are repaired before DNA replication starts. This fraction of repaired DNA is independent of the location of the mismatched nucleotide pair. However, mismatch correction occurs preferentially on the H strand of the heteroduplex DNA. The repair does not depend on a known phage coded function but requires the active bacterial genes mutU, mutH, mutS and probably mutL.

Base Sequence↗