P. C. Van Der Vliet

Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH.

We have studied promoter opening in assays reconstituted with purified RNA polymerase II and basal transcription factors. We found that creating a region of heteroduplex DNA around the start site of the adenovirus major late (AdML) promoter circumvents the requirement for TFIIE and TFIIH in transcription. The critical size and position of the heteroduplex region that alleviates the requirement for TFIIE and TFIIH is six nucleotides, from −4 to +2. Promoter opening was investigated directly with potassium permanganate (KMnO4), a chemical probe specific for single‐stranded thymidines. We found that KMnO4‐detectable opening of the AdML promoter requires the presence of the complete pre‐initiation complex, DBpolFEH, and that opening occurs in two discrete steps. First, dependent on ATP but prior to initiation, the −9 to +1 region becomes single‐stranded. Second, formation of the first phosphodiester bond results in expansion of the open region to the +8 position. Our results lead to a model in which the critical function of the TFIIH‐associated DNA helicases is to create a single‐stranded region. This gives RNA polymerase II access to the nucleotides of the template strand and allows expansion of the open region upon formation of the first phosphodiester bond.

The requirement for the basal transcription factor IIE is determined by the helical stability of promoter DNA.

The role of the basal transcription factor TFIIE was investigated in RNA polymerase II transcription reactions reconstituted with purified proteins. Using negatively supercoiled templates, which circumvent the requirement for TFIIH, we observed that transcription from the adenovirus major‐late (ML) core promoter is more dependent on TFIIE than transcription from the adenovirus E4 (E4) or mouse mammary tumor virus (MMTV) promoters. For all three promoters, an increase in the ionic strength of the reaction mixtures led to an increased dependence on TFIIE. Analysis of hybrid ML/MMTV promoters showed that the region encompassing the start site, from ‐10 to +10, dictates this dependence. Transcription from a relaxed E4 template with a pre‐melted ‐8 to +2 region was completely independent of both TFIIE and TFIIH. We propose that on negatively supercoiled templates TFIIE can facilitate promoter melting.

Early, current and past pet ownership: associations with sensitization, bronchial responsiveness and allergic symptoms in school children

Background Studies have suggested that early contact with pets may prevent the development of allergy and asthma.

The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with Oct-1/Oct-2 factors, leading to differential promoter activation.

We have shown previously that both octamer binding transcription factors, namely the ubiquitous Oct‐1 and the B cell‐specific Oct‐2A protein, can be enhanced in transcriptional activity by their association with the B cell‐specific coactivator protein Bob1, also called OBF‐1 or OCA‐B. Here we study the structural requirements for ternary complex formation of DNA‐Oct‐Bob1 and coactivation function of Bob1. In analogy to DNA‐bound transcription factors, Bob1 has a modular structure that includes an interaction domain (amino acids 1–65) and a C‐terminal domain (amino acids 65–256), both important for transcriptional activation. A mutational analysis has resolved a region of seven amino acids (amino acids 26–32) in the N‐terminus of Bob1 that are important for contacting the DNA binding POU domain of Oct‐1 or Oct‐2. In contrast to the viral coactivator VP16 (vmw65), which interacts with Oct‐1 via the POU homeosubdomain, Bob1 association with Oct factors requires residues located in the POU‐specific subdomain. Because the same residues are also involved in DNA recognition, we surmised that this association would affect the DNA binding specificity of the Oct‐Bob1 complex compared with free Oct factors. While Oct‐1 or Oct‐2 bind to a large variety of octamer sequences, Bob1 ternary complex formation is indeed highly selective and occurs only in a subset of these sequences, leading to the differential coactivation of octamer‐containing promoters. The results uncover a new level in selectivity that furthers our understanding in the regulation of cell type‐specific gene expression.

Amino-terminal domain of NF1 binds to DNA as a dimer and activates adenovirus DNA replication.

NF1 is a DNA‐binding protein involved in initiation of adenovirus DNA replication as well as in modulating the rate of transcription initiation of genes containing the sequence TGGCA. We show here that recombinant NF1 expressed via vaccinia virus is transported into the nucleus and binds to its cognate sequences with the same specificity as NF1 purified from HeLa cells. Furthermore, the recombinant NF1 forms oligomers in solution and binds as a dimer to palindromic as well as half‐site sequences. NF1 expressed via vaccinia virus stimulates the initiation of adenovirus replication in vitro. The N‐terminal 240 amino acids of the protein are sufficient for full DNA‐binding activity as well as stimulation of adenovirus replication. By analysis of several NF1 mutants translated in vitro, we also define the minimal DNA‐binding domain and localize the region responsible for DNA binding on the N‐terminal and for oligomerization on the C‐terminal side of this domain.

A precursor terminal protein-trinucleotide intermediate during initiation of adenovirus DNA replication: regeneration of molecular ends in vitro by a jumping back mechanism.

The adenovirus type 5 origin sequence starts with 3′ GTAGTA. Initiation of replication occurs by a protein priming mechanism in which the viral precursor terminal protein (pTP) is covalently linked to the first nucleotide of the nascent chain, a dCMP residue. This suggests that a pTP‐dCMP (pTP‐C) complex functions as an initiation intermediate. Employing a reconstituted replication system and both synthetic oligonucleotides and the natural TP‐DNA as templates, we show that pTP‐CAT rather than pTP‐C is an intermediate in initiation. By replicating oligonucleotide templates mutated at different positions and analyzing the product lengths, we observed that the GTA at positions 4‐6, rather than 1‐3, are used as a template for pTP‐CAT formation. Moreover, deletions of one or two nucleotides at the molecular ends were regenerated upon in vitro replication. Our results support a model in which the pTP‐CAT intermediate, synthesized opposite to positions 4‐6, jumps back to position 1 of the template to start elongation. In order to permit elongation, some base pairing between pTP‐CAT and template residues 1‐3 is required. This jumping‐back mechanism ensures the integrity of terminal sequences during replication of the linear genome.

Contactpoint analysis of the HeLa nuclear factor I recognition site reveals symmetrical binding at one side of the DNA helix.

Nuclear factor I (NFI) is a HeLa sequence‐specific DNA‐binding protein that is required for initiation of adenovirus (Ad) DNA replication and may be involved in the expression of several cellular genes. The interaction between NFI and its binding site on the Ad2 origin has been studied. Methylation interference and protection, u.v. irradiation of 5‐BrdU substituted DNA and ethylation interference revealed major groove contacts with G and T, and phosphate backbone contacts. Computer stereographics show that the contacts are located in two blocks showing dyad symmetry to each other and 22 out of 23 contacts are accessible from one side of the helix. Inversion of the NFI binding site did not change the NFI dependent stimulation of Ad2 DNA replication in a reconstituted system. All data are compatible with NFI binding as a dimer at one side of the DNA helix.

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.

Adenovirus DNA Replication

Studies on the replication of adenovirus DNA were initiated more than two decades ago and quickly led to a novel displacement model for DNA replication (Sussenbach et al. 1972). These studies were mainly performed using intact infected cells or isolated nuclei. It was only after the development of a system to study replication in vitro (Challberg and Kelly 1979) that detailed information could be obtained about the protein-priming mechanism for initiation and about the replication proteins. The last decade has been characterized by the discovery of transcription factors as participants in initiation (Nagata et al. 1982; Pruijn et al. 1986), by complete reconstitution of the system with purified recombinant proteins, and by structural information on some of the replication proteins. After the previous review in this series (Sussenbach and Van der Vliet 1983), several reviews on adenovirus replication have appeared (Kelly1984; Campbell 1986; Van der Vliet et al. 1988; Challberg Kelly 1989; Stillman 1989; Hay and Russell 1989; Van der Vliet 1990, 1991; Salas 1991; De Pamphilis 1993a).

POU proteins bend DNA via the POU-specific domain.

POU proteins constitute a family of ubiquitous as well as cell type‐specific transcription factors that share the conserved POU DNA binding domain. This domain consists of two distinct subdomains, a POU‐specific domain and a POU homeodomain, that are both required for high affinity sequence‐specific DNA binding. In a circular permutation assay, several POU proteins, including Oct‐1, Oct‐2A, Oct‐6 and Pit‐1, demonstrated a position dependent mobility of the protein‐DNA complexes, suggesting induction of DNA bending. This was confirmed by detection of relative bend direction, using pre‐bent DNA, and by enhanced ligase mediated cyclization. Bending was caused by interaction with the POU domain. By contrast, binding of the POU homeodomain did not distort the DNA structure, indicating that the POU‐specific domain confers DNA bending.