Peter C. van der Vliet

An expression profile for diagnosis of lymph node metastases from primary head and neck squamous cell carcinomas

Metastasis is the process by which cancers spread to distinct sites in the body. It is the principal cause of death in individuals suffering from cancer. For some types of cancer, early detection of metastasis at lymph nodes close to the site of the primary tumor is pivotal for appropriate treatment. Because it can be difficult to detect lymph node metastases reliably, many individuals currently receive inappropriate treatment. We show here that DNA microarray gene-expression profiling can detect lymph node metastases for primary head and neck squamous cell carcinomas that arise in the oral cavity and oropharynx. The predictor, established with an 82-tumor training set, outperforms current clinical diagnosis when independently validated. The 102 predictor genes offer unique insights into the processes underlying metastasis. The results show that the metastatic state can be deciphered from the primary tumor gene-expression pattern and that treatment can be substantially improved.

HELA NUCLEAR-PROTEIN RECOGNIZING DNA TERMINI AND TRANSLOCATING ON DNA FORMING A REGULAR DNA MULTIMERIC PROTEIN COMPLEX

Employing an exonuclease III protection assay we detected a protein in crude HeLa nuclear extracts binding, with apparent sequence specificity, to molecular ends of adenovirus type 2 (Ad2) DNA. This protein, designated nuclear factor IV (NFIV), was purified to homogeneity and was shown to be a hetero-dimer of 72,000 and 84,000 Mr. Binding to terminal Ad2 sequences was strongly enhanced by the presence of either of the sequence-specific DNA-binding proteins nuclear factor I and nuclear factor III. These proteins appeared to function as blockades for translocation of NFIV on DNA, thus producing apparent sequence specificity. In the absence of such a blockade, NFIV moved freely, without energy input, on any double-stranded DNA forming a regular DNA-multimeric protein complex as shown by methidiumpropyl EDTA footprinting and electron microscopy. Binding is completely dependent upon the presence of molecular ends. Evidence was obtained for a two-step mechanism in which termini are recognized by NFIV and used as a starting point for subsequent translocation. The possible functions of the protein in adenovirus DNA replication and in cellular processes requiring DNA termini are discussed.

An Adenovirus Type 5 Gene Function Required for Initiation of Viral DNA Replication

Adenovirus type 5 (Ad5) DNA replication was studied after infection of human or monkey cells with two DNA-negative temperature-sensitive mutants belonging to different complementation groups (H5ts125 and H5ts36). When infection was carried out at the permissive temperature (32°) followed by a shift to the nonpermissive temperature (39.5°) viral DNA synthesis in H5ts125-infected cells was reduced 90% within 1 hr after shift-up, while a decline in DNA synthesis in H5ts36-infected cells is only observed after 6 hr. Analysis of the various forms of DNA synthesized under conditions of inhibition showed a constant ratio of replicating to mature viral DNA for both mutants, while no accumulation of replicating molecules was observed. When H5ts125-infected cells were pulse-labeled with [3H]thymidine at 32 or 39.5° followed by a chase of the label at 39.5°, replicating DNA was converted into mature DNA at the same rate as in wild-type-infected cells. This indicates that chain propagation and termination could occur normally under nonpermissive conditions. The results of density labeling experiments performed at 39.5° are in agreement with an initiation block in H5ts125-infected cells at the nonpermissive temperature. It is concluded that the H5ts125 gene product and possibly also the H5ts36 gene product are required for the initiation of new rounds of replication. The potential role in initiation of the adenovirus-specific DNA binding protein, which is coded for by the H5ts125 gene, is discussed.

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.

Interaction of PC4 with melted DNA inhibits transcription

PC4 is a nuclear DNA‐binding protein that stimulates activator‐dependent class II gene transcription in vitro. Recent biochemical and X‐ray analyses have revealed a unique structure within the C‐terminal domain of PC4 that binds tightly to unpaired double‐stranded (ds)DNA. The cellular function of this evolutionarily conserved dimeric DNA‐binding fold is unknown. Here we demonstrate that PC4 represses transcription through this motif. Interaction with melted promoters is not required for activator‐dependent transcription in vitro. The inhibitory activity is attenuated on bona fide promoters by (i) transcription factor TFIIH and (ii) phosphorylation of PC4. PC4 remains a potent inhibitor of transcription in regions containing unpaired ds DNA, in single‐stranded DNA that can fold into two antiparallel strands, and on DNA ends. Our observations are consistent with a novel inhibitory function of PC4.

The Rep Protein of Adeno-Associated Virus Type 2 Interacts with Single-Stranded DNA-Binding Proteins That Enhance Viral Replication

ABSTRACT Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1) proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions.

POU domain transcription factors in embryonic development

One of the families of transcription factors implicated in developmental control of gene expression is that of the POU domain proteins [1–5]. These transcription factors contain a region of sequence similarity in their DNA binding domains, which is referred to as the POU domain, as it was initially identified in the mammalian transcription factors Pit-1, Oct-1, and Oct-2, and in the nematode unc-86 gene product [1], see Figure 1. Since the initial identification of the POU domain, the list of identified POU box genes has considerably expanded [4–7]; so far, 20 different genes have been cloned from vertebrate cDNA and genomic libraries [7], whereas four and three genes have been isolated from respectively the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans [5, 7]. POU domain proteins are interesting transcriptional regulators from several points of view. Firstly, because the mechanism by which these factors bind to DNA appears to involve tethering of two independently folded DNA binding helix-turn-helix motifs. This mechanism has been reviewed recently [5], and will not be discussed extensively here. Secondly, research on transcriptional regulation by POU factors has provided new insights as to how cell type-specific gene expression is regulated, and how this control of gene expression results in differentiated cellular phenotypes during development. The levels at which POU domain proteins are regulated will be discussed, as well as the biological function of POU domain transcription factors. The last topic is about redundant, divergent and emergent functions [8] of POU domain proteins that result from co-expression of their genes in one cell type. The POU domain: a bipartite DNA binding domain

Multimerization of the adenovirus DNA‐binding protein is the driving force for ATP‐independent DNA unwinding during strand displacement synthesis

In contrast to other replication systems, adenovirus DNA replication does not require a DNA helicase to unwind the double‐stranded template. Elongation is dependent on the adenovirus DNA‐binding protein (DBP) which has helix‐destabilizing properties. DBP binds cooperatively to single‐stranded DNA (ssDNA) in a non‐sequence‐specific manner. The crystal structure of DBP shows that the protein has a C‐terminal extension that hooks on to an adjacent monomer which results in the formation of long protein chains. We show that deletion of this C‐terminal arm results in a monomeric protein. The mutant binds with a greatly reduced affinity to ssDNA. The deletion mutant still stimulates initiation of DNA replication like the intact DBP. This shows that a high affinity of DBP for ssDNA is not required for initiation. On a single‐stranded template, elongation is also observed in the absence of DBP. Addition of DBP or the deletion mutant has no effect on elongation, although both proteins stimulate initiation on this template. Strand displacement synthesis on a double‐stranded template is only observed in the presence of DBP. The mutant, however, does not support elongation on a double‐stranded template. The unwinding activity of the mutant is highly reduced compared with intact DBP. These data suggest that protein chain formation by DBP and high affinity binding to the displaced strand drive the ATP‐independent unwinding of the template during adenovirus DNA replication.

The Oct-1 POU Homeodomain Stabilizes the Adenovirus Preinitiation Complex via a Direct Interaction with the Priming Protein and Is Displaced when the Replication Fork Passes

Initiation of adenovirus DNA replication is strongly enhanced by two cellular transcription factors, NFI and Oct-1, which bind to the auxiliary origin and tether the viral precursor terminal protein-DNA polymerase (pTP·pol) complex to the core origin. NFI acts through a direct contact with the DNA polymerase, but the mode of action of Oct 1 is unknown. Employing glutathione S-transferase-POU pull-down assays and protein affinity chromatography, we have established that the POU domain contacts pTP rather than pol. The POU homeodomain is responsible for this interaction. The protein-protein contacts lead to increased binding of pTP-pol to the core origin, which is caused by a reduced off-rate. The enhanced formation of a pTP·pol·POU complex on the origin correlates with stimulation of replication. Using an immobilized replication system, we have studied the kinetics of dissociation of the Oct-1 POU domain during replication. In contrast to NFI, which dissociates very early in initiation, Oct-1 dissociates only when the binding site is rendered single-stranded upon translocation of the replication fork. Our data indicate that NFI and Oct-1 enhance initiation synergistically by touching different targets in the preinitiation complex and dissociate independently after initiation.

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 inK m 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.