Biomedical subjects

P C van der Vliet

Publications and source records attributed to P C van der Vliet.

At least 19 recordsLinked to original sources

PubMed2003

DNA binding properties of the adenovirus DNA replication priming protein pTP.

The precursor terminal protein pTP is the primer for the initiation of adenovirus (Ad) DNA replication and forms a heterodimer with Ad DNA polymerase (pol). Pol can couple dCTP to pTP directed by the fourth nucleotide of the viral genome template strand in the absence of other replication proteins, which suggests that pTP/pol binding destabilizes the origin or stabilizes an unwound state. We analyzed the contribution of pTP to pTP/pol origin binding using various DNA oligonucleotides. We show that two pTP molecules bind cooperatively to short DNA duplexes, while longer DNA fragments are bound by single pTP molecules as well. Cooperative binding to short duplexes is DNA sequence independent and most likely mediated by protein/protein contacts. Furthermore, we observed that pTP binds single-stranded (ss)DNA with a minimal length of approximately 35 nt and that random ssDNA competed 25-fold more efficiently than random duplex DNA for origin binding by pTP. Remarkably, short DNA fragments with two opposing single strands supported monomeric pTP binding. pTP did not stimulate, but inhibited strand displacement by the Ad DNA binding and unwinding protein DBP. These observations suggest a mechanism in which the ssDNA affinity of pTP stabilizes Ad pol on partially unwound origin DNA.

Binding Sites↗

PubMed2003

Adenovirus DNA replication: protein priming, jumping back and the role of the DNA binding protein DBP.

The adenovirus (Ad) genome is a linear double-stranded (ds) molecule containing about 36 kilobase pairs. At each end of the genome an approximately 100 base pair (bp) inverted terminal repeat (ITR) is found, the exact length depending on the serotype. To the 5'-end of each ITR, a 55-kDa terminal protein (TP) is covalently coupled. The Ad DNA replication system was one of the first replication systems that could be reconstituted in vitro (Challberg and Kelly 1979). The system requires three virally encoded proteins: precursor TP (pTP), DNA polymerase (Pol) and the DNA binding protein (DBP). In addition, three stimulating human cellular proteins have been identified. These are the transcription factors NFI (Nagata et al. 1982) and Oct-1 (Pruijn et al. 1986) and the type I topoisomerase NFII (Nagata et al. 1983). Ad DNA replication uses a protein primer for replication initiation. The transition from initiation to elongation is marked by a jumping back mechanism (King and van der Vliet 1994), followed by elongation. In order to elongate DBP is required. In this review we discuss the roles of DBP during initiation and elongation and we relate biochemical data on the jumping back mechanism used by Ad Pol to the recently solved crystal structure of a Pol alpha-like replication complex (Franklin et al. 2001). We comment on the conditions and possible functions of jumping back and propose a model to describe the jumping back mechanism.

Adenoviridae↗

PubMed2002

Recruitment of the priming protein pTP and DNA binding occur by overlapping Oct-1 POU homeodomain surfaces.

The human transcription factor Oct-1 can stimulate transcription from a variety of promoters by interacting with the coactivators OBF-1/OCA-B/BOB-1, SNAP190 and VP16. These proteins contact Oct-1 regions different from the DNA binding surface. Oct-1 also stimulates the DNA replication of adenovirus through its DNA binding site in the origin. The Oct-1 POU homeodomain (POUhd) binds the adenovirus precursor terminal protein pTP, which serves as the protein primer of DNA replication and recruits pTP to the origin. To map the interaction with pTP at the POUhd surface, we screened a library of randomly mutated POU domains and identified mutations that interfered with pTP interaction and DNA replication stimulation. These mutants clustered at a surface different from those recognized by OBF-1, SNAP190 and VP16. Unexpectedly, the pTP binding region largely overlapped with the DNA binding surface of POUhd. In agreement with this, pTP binding and DNA binding were mutually exclusive. We propose a model to reconcile pTP recruitment and DNA binding by Oct-1.

Adenoviridae↗

PubMed2001

The (I/Y)XGG motif of adenovirus DNA polymerase affects template DNA binding and the transition from initiation to elongation.

Adenovirus DNA polymerase (Ad pol) is a eukaryotic-type DNA polymerase involved in the catalysis of protein-primed initiation as well as DNA polymerization. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-directed mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex DNA. Three mutant polymerases required higher nucleotide concentrations for effective polymerization and showed higher exonuclease activity on double-stranded DNA. These observations suggest a local destabilization of DNA substrate at the polymerase active site. In agreement with this, the mutant polymerases showed reduced initiation activity and increased K(m)(app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while capable of elongating on the primer-template DNA, failed to elongate after protein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and ceased completely after formation of the precursor terminal protein-trinucleotide (pTP-CAT) initiation intermediate. Our results suggest that residues in the conserved motif (I/Y)XGG in Ad pol are involved in binding the template strand in the polymerase active site and play an important role in the transition from initiation to elongation.

Adenoviridae↗

PubMed2001

Combined pituitary hormone deficiency caused by compound heterozygosity for two novel mutations in the POU domain of the Pit1/POU1F1 gene.

The POU homeodomain containing transcriptional activator POU1F1, formerly called Pit1 or GHF-1, is required for the embryological determination and postnatal secretory function of the GH-, PRL-, and TSH-producing cells in the anterior pituitary. Several mutations in the gene encoding POU1F1 have been described, resulting in a syndrome of combined pituitary hormone deficiency involving these three hormones. Most of the patients with this phenotype have either a dominant negative mutation in codon 271 (R271W) or are homozygous for a recessive mutation in the POU1F1 gene; to date only one case has been reported with compound heterozygosity for two point mutations. Here, we describe a boy with severe deficiencies of GH, PRL, and TSH who had compound heterozygosity for two novel point mutations in the POU1F1 gene: a 1-bp deletion frameshift mutation (747delA), the first one described to date in this gene, which leads to a nonfunctional truncated protein lacking the entire DNA recognition helix of the POU homeodomain, and a missense mutation in the C-terminal end of the fourth alpha-helix of the POU-specific domain (W193R),which causes a 500-fold reduction in the ability to bind to DNA and activate transcription.

Cell Line↗

PubMed2000

The formation of a flexible DNA-binding protein chain is required for efficient DNA unwinding and adenovirus DNA chain elongation.

The adenovirus DNA-binding protein (DBP) binds cooperatively to single-stranded DNA (ssDNA) and stimulates both initiation and elongation of DNA replication. DBP consists of a globular core domain and a C-terminal arm that hooks onto a neighboring DBP molecule to form a stable protein chain with the DNA bound to the internal surface of the chain. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. As shown by x-ray diffraction of different crystal forms of the C-terminal domain, the C-terminal arm can adopt different conformations, leading to flexibility in the protein chain. This flexibility is a function of the hinge region, the part of the protein joining the C-terminal arm to the protein core. To investigate the function of the flexibility, proline residues were introduced in the hinge region, and the proteins were purified to homogeneity after baculovirus expression. The mutant proteins were still able to bind ss- and double-stranded DNA with approximately the same affinity as wild type, and the binding to ssDNA was found to be cooperative. All mutant proteins were able to stimulate the initiation of DNA replication to near wild type levels. However, the proline mutants could not support elongation of DNA replication efficiently. Even the elongation up to 26 nucleotides was severely impaired. This defect was also seen when DNA unwinding was studied. Binding studies of DBP to homo-oligonucleotides showed an inability of the proline mutants to bind to poly(dA)(40), indicating an inability to adapt to specific DNA conformations. Our data suggest that the flexibility of the protein chain formed by DBP is important in binding and unwinding of DNA during adenovirus DNA replication. A model explaining the need for flexibility of the C-terminal arm is proposed.

Adenoviridae↗

PubMed2000

A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation.

Proliferating cell nuclear antigen is best known as a DNA polymerase accessory protein but has more recently also been shown to have different functions in important cellular processes such as DNA replication, DNA repair, and cell cycle control. PCNA has been found in quaternary complexes with the cyclin kinase inhibitor p21 and several pairs of cyclin-dependent protein kinases and their regulatory partner, the cyclins. Here we show a direct interaction between PCNA and Cdk2. This interaction involves the regions of the PCNA trimer close to the C termini. We found that PCNA and Cdk2 form a complex together with cyclin A. This ternary PCNA-Cdk2-cyclin A complex was able to phosphorylate the PCNA binding region of the large subunit of replication factor C as well as DNA ligase I. Furthermore, PCNA appears to be a connector between Cdk2 and DNA ligase I and to stimulate phosphorylation of DNA ligase I. Based on our results, we propose the model that PCNA brings Cdk2 to proteins involved in DNA replication and possibly might act as an "adaptor" for Cdk2-cyclin A to PCNA-binding DNA replication proteins.

CDC2-CDC28 Kinases↗

PubMed1999

Mechanism of DNA replication in eukaryotic cells: cellular host factors stimulating adenovirus DNA replication.

Replication of adenovirus (Ad) DNA depends on interactions between three viral and three cellular proteins. Human transcription factors NFI and Oct-1 recruit the Ad DNA polymerase to the origin of DNA replication as a complex with the Ad protein primer pTP. High affinity and specificity DNA binding to recognition sites in this origin by the transcription factors stimulate and stabilize pre-initiation complex formation to compensate for the low binding specificity of the pTP/pol complex. In this review, we discuss the properties of NFI and Oct-1 and the mechanism by which they enhance initiation of DNA replication. We propose a model that describes the dynamics of initiation and elongation as well as the assembly and disassembly of the pre-initiation complex.

Adenoviridae↗

PubMed1999

Identification of the single-stranded DNA binding surface of the transcriptional coactivator PC4 by NMR.

The C-terminal domain of the eukaryotic transcriptional cofactor PC4 (PC4CTD) is known to bind with nanomolar affinity to single-stranded (ss)DNA. Here, NMR is used to study DNA binding by this domain in more detail. Amide resonance shifts that were observed in a 1H15N-HSQC-monitored titration of 15N-labeled protein with the oligonucleotide dT18 indicate that binding of the nucleic acid occurs by means of two anti-parallel channels that were previously identified in the PC4CTD crystal structure. The beta-sheets and loops that make up these channels exhibit above average flexibility in the absence of ssDNA, which is reflected in higher values of T1rho, reduced heteronuclear nuclear Overhauser effects and faster deuterium exchange rates for the amides in this region. Upon ssDNA binding, this excess flexibility is significantly reduced. The binding of ssDNA by symmetry-related channels reported here provides a structural rationale for the preference of PC4CTD for juxtaposed single-stranded regions (e.g. in heteroduplexes) observed in earlier work.

Binding Sites↗

PubMed1998

Sequences flanking the E-box contribute to cooperative binding by c-Myc/Max heterodimers to adjacent binding sites.

Previously, we have shown that c-Myc/Max heterodimers, bind cooperatively to the two adjacent, canonical E-boxes (CACGTG) located in the rat ornithine decarboxylase (ODC) gene. In order to study this in more detail, we changed the length of the linker that separates the two E-boxes, as well as their flanking sequences. We found that high affinity, cooperative binding requires a minimal linker length of 1-4 bp and that the binding affinity is influenced by E-box flanking sequences. Binding to the c-Myc responsive element of prothymosin alpha, containing both a canonical and a noncanonical E-box (CAAGTG) was also studied. As shown by DNAseI footprinting analysis, only the canonical E-box is bound by c-Myc/Max and c-Max/Max dimers. Replacing the noncanonical site with a canonical E-box only partially restored high affinity, cooperative binding. By making hybrid fragments between ODC and prothymosin alpha, we found that nucleotides in the linker between the E-boxes influence the affinity of c-Myc/Max heterodimers. Taken together, our results show that E-box sequences and sequences in the linker separating both E-boxes influence cooperative, high affinity binding by c-Myc/Max dimers.

Animals↗

PubMed1998

ATP-independent DNA unwinding by the adenovirus single-stranded DNA binding protein requires a flexible DNA binding loop.

The adenovirus DNA binding protein (DBP) binds cooperatively to single-stranded (ss) DNA and stimulates both initiation and elongation of DNA replication. DBP forms protein filaments via a C-terminal arm that hooks into a neighbouring molecule. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. Another conserved part of DBP forms an unstructured flexible loop that is probably directly involved in contacting DNA. By making appropriate deletion mutants that do not distort the overall DBP structure, the influence of the C-terminal arm and the flexible loop on the kinetics of ssDNA binding and on DNA replication was studied. Employing surface plasmon resonance we show that both parts of the protein are required for high affinity binding. Deletion of the C-terminal arm leads to an extremely labile DBP-ssDNA complex indicating the importance of multimerization. The flexible loop is also required for optimal stability of the DBP-ssDNA complex, providing additional evidence that this region forms part of the ssDNA-binding surface of DBP. Both deletion mutants are still able to stimulate initiation of DNA replication but are defective in supporting elongation, which may be caused by the fact that both mutants have a reduced DNA unwinding activity. Surprisingly, mixtures containing both mutants do stimulate elongation. Mixing the purified mutant proteins leads to the formation of mixed filaments that have a higher affinity for ssDNA than homogeneous mutant filaments. These results provide evidence that the C-terminal arm and the flexible loop have distinct functions in unwinding during replication. We propose the following model for ATP-independent DNA unwinding by DBP. Multimerization via the C-terminal arm is required for the formation of a protein filament that saturates the displaced strand. A high affinity of a DBP monomer for ssDNA and subsequent local destabilization of the replication fork requires the flexible loop.

Adenosine Triphosphate↗

PubMed1998

High-affinity DNA binding by the C-terminal domain of the transcriptional coactivator PC4 requires simultaneous interaction with two opposing unpaired strands and results in helix destabilization.

The general transcriptional cofactor PC4 enhances transcription from various promoters and functions with a wide range of transcriptional activators. Earlier studies have suggested that this enhancement originates mostly from stabilization of the TATA-box/TFIID/TFIIA complex by simultaneous interaction of PC4 with transactivation domains of upstream-binding factors and the basal factor TFIIA. However, the C-terminal half of the protein also has been shown to exhibit substantial ssDNA binding properties, to which as yet no clear function has been assigned. We have investigated the interaction of this domain with various DNA structures and report that high-affinity binding, characterized by an equilibrium dissociation constant in the nanomolar range, requires either a heteroduplex containing a minimum of about eight mismatches, or alternatively a single-stranded DNA molecule consisting of 16 to 20 nucleotides. Furthermore, both juxtaposed single strands of a heteroduplex are protected by the C-terminal domain of PC4 in DNase I footprinting experiments, whereas the double-stranded regions do not appear to be contacted. We conclude from these observations that the role of PC4 ssDNA binding is likely to involve simultaneous interaction with opposing strands in internally melted duplexes, or the induction of a pronounced distortion in the local structure of ssDNA that results in a similar juxtaposed arrangement of single strands. In addition, we have observed that both the PC4 C-terminal domain and the intact PC4 destabilize dsDNA and we discuss the possible involvement of PC4 in promoter opening and other strand displacement events.

Binding Sites↗

PubMed1998

Non-cell autonomous induction of apoptosis and loss of posterior structures by activation domain-specific interactions of Oct-1 in the Xenopus embryo.

Oct-1, a member of the POU family of transcription factors, is expressed at relatively high levels in ectodermal and mesodermal cell lineages during early Xenopus embryogenesis (Veenstra et al, 1995). Here we show that overexpression of Oct-1 induces programmed cell death concomitant with the loss of the posterior part of the body axis. Truncated Oct-1 variants, missing either the C-terminal or N-terminal trans-activation domain, exhibit a different capacity to cause such developmental defects. Oct-1-induced cell death is rescued in unilaterally injected embryos by non-injected cells, indicative of the non-cell autonomous character of the developmental effects of Oct-1. This was confirmed by marker gene analysis, which showed a significant decrease in brachyury expression, suggesting that Oct-1 interferes with an FGF-type signalling pathway.

Animals↗

PubMed1997

Cloning of the cDNA for the TATA-binding protein-associated factorII170 subunit of transcription factor B-TFIID reveals homology to global transcription regulators in yeast and Drosophila.

The human transcription factor B-TFIID is comprised of TATA-binding protein (TBP) in complex with one TBP-associated factor (TAF) of 170 kDa. We report the isolation of the cDNA for TAFII170. By cofractionation and coprecipitation experiments, we show that the protein encoded by the cDNA encodes the TAF subunit of B-TFIID. Recombinant TAFII170 has (d)ATPase activity. Inspection of its primary structure reveals a striking homology with genes of other organisms, yeast MOT1, and Drosophila moira, which belongs to the Trithorax group. Both homologs were isolated in genetic screens as global regulators of pol II transcription. This supports our classification of B-TFIID as a pol II transcription factor and suggests that specific TBP-TAF complexes perform distinct functions during development.

Adenosine Triphosphatases↗

PubMed1997

Dissociation of the protein primer and DNA polymerase after initiation of adenovirus DNA replication.

Initiation of adenovirus DNA replication occurs by a jumping back mechanism in which the precursor terminal priming protein (pTP) forms a pTP.trinucleotide complex (pTP.CAT) catalyzed by the viral DNA polymerase (pol). This covalent complex subsequently jumps back 3 bases to permit the start of chain elongation. Before initiation, pTP and pol form a tight heterodimer. We investigated the fate of this pTP.pol complex during the various steps in replication. Employing in vitro initiation and elongation on both natural viral templates and synthetic oligonucleotides followed by glycerol gradient separation of the reaction products, we established that pTP and pol are separated during elongation. Whereas pTP.C and pTP. CA were still bound to the polymerase, after the formation of pTP. CAT 60% of the pTP.pol complex had dissociated. Dissociation coincides with a change in sensitivity to inhibitors and in Km for dNTPs, suggesting a conformational change in the polymerase, both in the active site and in the pTP interaction domain. In agreement with this, the polymerase becomes a more efficient enzyme after release of the pTP primer. We also investigated whether the synthesis of a pTP initiation intermediate is confined to three nucleotides. Employing synthetic oligonucleotide templates with a sequence repeat of two nucleotides (GAGAGAGA ... instead of the natural GTAGTA ... ) we show that G5 rather than G3 is used to start, leading to a pTP. tetranucleotide (CTCT) intermediate that subsequently jumps back. This indicates flexibility in the use of the start site with a preference for the synthesis of three or four nucleotides during initiation rather than two.

Adenoviridae↗

PubMed1997

Processive proofreading by the adenovirus DNA polymerase. Association with the priming protein reduces exonucleolytic degradation.

By using a baculovirus expression system, the adenovirus (Ad) DNA polymerase was purified to homogeneity and shown to display a 3'-->5'exonuclease activity which is coupled to the polymerase activity. On a partial duplex structure the exonuclease activity had a marked preference for excision of a mismatched versus a matched 3'-terminus, which enables the Ad DNA polymerase to act as a proofreading enzyme. On single-stranded DNA the exonuclease action is distributive, but during replication removal of mismatched nucleotides and the switch to synthesis occurs without dissociation of the polymerase from the template. When the Ad DNA polymerase is bound to the precursor terminal protein, the rate of exonucleolysis was four times slower. Moreover, degradation could not proceed as far as with the free Ad polymerase, indicating also a qualitative difference. These results suggest a reduced proofreading capacity of the precursor terminal protein-polymerase complex, which might affect the initial stages of DNA replication.

Adenoviridae↗

PubMed1997

Linker length and composition influence the flexibility of Oct-1 DNA binding.

POU domain transcription factors have two separate helix-turn-helix DNA-binding subdomains, the POU homeodomain (POUhd) and the POU-specific domain (POUs). Each subdomain recognizes a specific subsite of 4 or 5 bp in the octamer recognition sequence. The Oct-1 POU subdomains are connected by a 23 amino acid unstructured linker region. To investigate the requirements for the linker and its role in DNA recognition, we constructed POU domains in which the subdomains are connected with linkers varying in length between 2 and 37 amino acids. Binding to the natural octamer site required a minimal linker length of between 10 and 14 amino acids. A POU domain with an eight amino acid linker, however, had a high affinity for a site in which the POUs recognition sequence was inverted. Computer modelling shows that inversion of the POUs subdomain shortens the distance between the subdomains sufficiently to enable an eight amino acid linker to bridge the distance. DNase I footprinting as well as mutation of the POUs-binding site confirms the inverted orientation of the POUs domain. Switching of the POUs and POUhd subdomains and separation by 3 bp leads to a large distance which could only be bridged effectively by a long 37 amino acid linker. In addition to linker length, mutation of a conserved glutamate residue in the linker affected binding. As shown by surface plasmon resonance measurements, this was caused by a decrease in the on-rate. Our data indicate that there are both length and sequence requirements in the linker region which allow flexibility leading to selective binding to differently spaced and oriented subsites.

Amino Acid Sequence↗

PubMed1997

c-Myc/Max heterodimers bind cooperatively to the E-box sequences located in the first intron of the rat ornithine decarboxylase (ODC) gene.

The oncoprotein c-Myc plays an important role in cell proliferation, transformation, inhibition of differentiation and apoptosis. These functions most likely result from the transcription factor activity of c-Myc. As a heterodimer with Max, the c-Myc protein binds to the E-box sequence (CACGTG), which is also recognized by USF dimers. In order to test differences in target gene recognition of c-Myc/Max, Max and USF dimers, we compared the DNA binding characteristics of these proteins in vitro using vaccinia viruses expressing full-length c-Myc and Max proteins. As expected, purified c-Myc/max binds specifically to a consensus E-box. The optimal conditions for DNA binding by either c-Myc/Max, Max or USF dimers differ with respect to ionic strength and Mg2+ ion concentration. Most interestingly, the c-Myc/Max complex binds with a high affinity to its natural target, the rat ODC gene, which contains two adjacent, consensus E-boxes. High affinity binding results from teh ability of c-Myc/Max dimers to bind cooperatively to these E-boxes. We propose that differential cooperative binding by E-box binding transcription factors could contribute to target gene specificity.

Amino Acid Sequence↗