Multiple cellular factors bind to cis-regulatory elements found inboard of the 5' palindrome of minute virus of mice.

Previous genetic analysis of the DNA replication of minute virus of mice (MVM) minigenomes suggested that specific elements, A (nucleotides [nt] 4489 to 4636) and B (nt 4636 to 4695), found inboard of the 5' palindrome are required for efficient MVM DNA replication (P. Tam and C. R. Astell, Virology 193:812-824, 1993). In this report, we show that two MVM RsaI restriction fragments (RsaI A [nt 4431 to 4579] and RsaI B [nt 4579 to 4662]) are able to activate DNA replication of an MVM minigenome containing deletions of both elements A and B. We also show that sequences inboard of the right palindrome are able to activate replication of minigenomes containing two left termini. In order to investigate the importance of the RsaI fragments, we demonstrate the presence of a number of sequence-specific DNA-protein interactions by electrophoretic mobility shift assays. After partial fractionation of A9 nuclear extracts, DNase I footprinting analysis was used to determine the binding sites for MVM replication factor (MRF) B5. MRF B5 protects two distinct regions (sites I and II) of the RsaI B probe from DNase I digestion. Competition f electrophoretic mobility shift assays with synthetic oligonucleotides corresponding to sites I and II suggest that MRF B5 is composed of two factors, MRF B3 and MRF B4, which bind DNA independently in a sequence-specific manner. It may be possible that these replication factors are proteins which are able to transactivate MVM DNA replication and hence are accessory replication factors.     

The DNA-binding properties of polyomavirus large T antigen are altered by ATP and other nucleotides.

We have examined the influence of ATP on the DNA-binding properties of polyomavirus large T antigen (Py TAg). Utilizing nitrocellulose filter binding, DNase I footprinting, and gel mobility shift assays, we observed that ATP increased Py TAg binding to DNA fragments containing either all Py TAg-binding sites (whole origin) or those sites within (core origin) or adjacent to (early) the origin of replication. Even nonspecific binding to DNA fragments lacking Py TAg-binding sites was increased somewhat by ATP. Binding to the core origin was increased to a greater extent than binding to other DNA fragments tested. Gel band mobility shift assays revealed that ATP increased the production of core origin-specific Py TAg-DNA complexes of high molecular weight. ATP stimulation depended on the presence of MgCl2. Other nucleotides and nonhydrolyzable ATP analogs also increased Py TAg binding to the core origin but to various degrees: ATP, dATP, 5'-adenylyl imidodiphosphate (AMPPNP) greater than 5'-adenylyl methylenediphosphate (AMPPCP) greater than dCTP greater than UTP greater than TTP. GTP and dGTP did not increase DNA binding by Py TAg. The rates of association and disassociation of Py TAg with all the DNA fragments were altered by the presence of ATP. DNase I footprinting showed that ATP extensively extended the region protected within the core origin and also produced a distinctive DNase I-hypersensitive site on the late strand at nucleotides 5255 to 5262 (TTACTATG).     

Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000.

Plasmid pAL5000 from Mycobacterium fortuitum encodes two proteins necessary for replication: RepA (307 amino acid residues) and RepB (119 residues). A single RNA species encoding these proteins was characterized, and its 5' end was defined. The proteins were expressed as maltose-binding protein fusions in Escherichia coli. The RepB protein was shown in vitro to bind specifically to a previously defined 435-bp region of pAL5000 containing the origin of replication (ori). The precise RepB binding sites were defined by DNase I footprinting experiments. RepB binds to two motifs in the ori region: a high-affinity site within its own promoter region, implying autoregulation of its expression, and a low-affinity site further upstream, presumably the origin of replication itself. The binding to the latter motif seems to occur on one DNA strand only. The high-affinity binding site contains several palindromic sequences. Gel retardation assays were performed with the different binding sites as templates, and the binding constant to each site was estimated from protein titrations. This is the first molecular dissection of mycobacterial DNA-binding proteins and their interactions with their targets.     

Biochemical analysis of a DNA replication origin in the archaeon Aeropyrum pernix

We have characterised the interaction of the Aeropyrum pernix origin recognition complex proteins (ORC1 and ORC2) with DNA using DNase I footprinting. Each protein binds upstream of its respective gene. However, ORC1 protein alone interacts more tightly with an additional region containing multiple origin recognition box (ORB) sites that we show to be a replication origin. At this origin, there are four ORB elements disposed either side of an A+T-rich region. An ORC1 protein dimer binds at each of these ORB sites. Once all four ORB sites have bound ORC1 protein, there is a transition to a higher-order assembly with a defined alteration in topology and superhelicity. Furthermore, after this transition, the A+T-rich region becomes sensitive to digestion by DNase I and P1 nuclease, revealing that the transition promotes distortion of the DNA in this region, presumably as a prelude to loading of MCM helicase.     

The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions.

The repressible acid phosphatase gene PHO5 of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHO5 gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHO5 promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PHO4 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASp1) and the other located at -239 to -262 (UASp2). Exonuclease III footprint experiments revealed stops at -349 and -368 (UASp1) as well as at -245 and -260 (UASp2). Gel retardation assays with the PHO2 protein revealed a binding region that lay between the two PHO4-binding sites. DNase I footprint analysis suggested a PHO2-binding site covering the region between -277 and -296.     

Herpes simplex virus origin-binding protein (UL9) loops and distorts the viral replication origin.

To investigate the role of the herpes simplex virus origin-binding protein (UL9) in the initiation of DNA replication, we have examined the effect of UL9 binding on the structure of the viral origin of replication. UL9 loops and alters the DNA helix of the origin regardless of the phasing of the binding sites. DNase I and micrococcal nuclease footprinting show that UL9 binds two sites in the origin and loops the AT-rich DNA between them independent of the topology of the DNA. KMnO4 and dimethyl sulfate footprinting further show that UL9 alters the DNA helix in the AT region. In contrast to the looping reaction, however, helical distortion requires the free energy of supercoiled DNA. UL9 also loops and distorts the origin DNA of a replication-defective mutant with a 6-bp insertion in the AT region. Because the helical distortion of this mutant DNA is different from that of functional origins, we conclude that an imperfect tertiary structure of the mutant DNA may contribute to its loss of replication function.     

Two nearby sites bind zen protein independently

Summary The region upstream from the zerknullt (zen) gene contains three sites that specifically bind the zen protein product of the gene. Evidence for these binding sites was obtained by the filter binding technique and the DNase footprinting technique. The filter binding technique was used to scan various segments of DNA for the presence of possible specific binding sites. Segments that were selectively retained by the filter binding technique invariably contained one or more specific binding sites according to the DNase footprinting technique. Two of the zen protein binding sites were spaced only 30 base pairs apart. These sites could be separated without any loss in their specific binding properties. It is concluded that these two sites function independently in the binding of zen protein.     

Gibberellin treatment stimulates nuclear factor binding to the gibberellin response complex in a barley alpha-amylase promoter.

The promoters of a majority of cereal alpha-amylase genes contain three highly conserved sequences (gibberellin response element, box I, and pyrimidine box). Recent studies have demonstrated the functional importance of four regions that either coincide with or are immediately proximal to these three conserved elements as well as an upstream Opaque-2 binding sequence. In this study, we describe the characterization of nuclear protein factors from barley aleurone layers whose binding activity toward gibberellin response complex sequences from the barley low-pl alpha-amylase gene (Amy32b) promoter is stimulated by gibberellin A3 (GA3) treatment. Barley proteins isolated from crude nuclear extracts prepared from aleurone layers incubated with or without GA3 were fractionated by anion exchange fast protein liquid chromatography and studied using band shift assays, sequence-specific competitions, and DNase I footprinting. A GA3-dependent binding activity eluting at 210 mM KCl was shown to bind specifically to the gibberellin response element and the closely associated box I. DNase I footprinting with the proteins in this fraction indicated interactions with sequences in the gibberellin response element and box I. A second DNA binding activity eluting at 310 mM KCl was present constitutively in extracts prepared from tissues incubated both in the absence and in the presence of hormone. Proteins in this fraction were able to bind to many DNA sequences and, in general, were largely nonspecific. DNase I footprinting with the proteins in this fraction indicated a large area of protection with a single unoccupied region located at the 3' end of box I. The possible function of such an activity in hormone regulation of the alpha-amylase genes is discussed.     

Purification and characterization of the Ner repressor of bacteriophage Mu

The Ner protein of bacteriophage Mu acts as a lambda cro-like negative regulator of the phage's early (transposase) operon. Using the band retardation assay to monitor ner-operator-specific DNA-binding activity, the 8 kDa Ner protein was purified to homogeneity. DNase I footprinting revealed that the purified protein bound and protected a specific DNA operator that contains two 12 bp sites with the consensus sequence 5'-ANPyTAPuCTAAGT-3', separated by a 6 bp spacer region. Moreover, regions corresponding to a turn of the DNA helix flanking these 12 bp repeats are also protected by Ner. Unlike the functionally similar lambda cro protein, gel filtration experiments show that the native molecular mass of Mu Ner to be approx. 8 kDa. These results, plus the pattern of DNase I protection, suggest that the protein may bind as a monomer to each of its specific DNA substrates.     

Identification of cellular factors that bind specifically to the Epstein-Barr virus origin of DNA replication.

The specific binding of HeLa cell factors to DNA sequences at the Epstein-Barr virus (EBV) latent origin of DNA replication was detected by gel shift experiments and DNase I footprinting analysis. These cellular proteins protected at least five discrete regions of the DNA replication origin. The viral protein required for EBV plasmid replication, EBV nuclear antigen 1 (EBNA-1), binds to specific sequences within the origin region. The HeLa cell proteins competed with EBNA-1 for binding to EBV origin DNA in vitro, leading to the possibility that these cellular proteins regulate EBV DNA replication by displacing EBNA-1 at the origin sites.     

Methidiumpropyl-EDTA.Fe(II) and DNase I footprinting report different small molecule binding site sizes on DNA.

DNase I and MPE.Fe (II) footprinting both employ partial cleavage of ligand-protected DNA restriction fragments and Maxam-Gilbert sequencing gel methods of analysis. One method utilizes the enzyme, DNase I, as the DNA cleaving agent while the other employs the synthetic molecule, methidium-propyl-EDTA (MPE). For actinomycin D, chromomycin A3 and distamycin A, DNase I footprinting reports larger binding site sizes than MPE.Fe (II). DNase I footprinting appears more sensitive for weakly bound sites. MPE.Fe (II) footprinting appears more accurate in determining the actual size and location of the binding sites for small molecules on DNA, especially in cases where several small molecules are closely spaced on the DNA. MPE.Fe (II) and DNase I report the same sequence and binding site size for lac repressor protein on operator DNA.     

Interaction of minor groove binding ligands with long AT tracts.

We have used quantitative DNase I footprinting to examine the ability of distamycin and Hoechst 33258 to discriminate between different arrangements of AT residues, using synthetic DNA fragments containing multiple blocks of (A/T)6or (A/T)10in identical sequence environments. Previous studies have shown that these ligands bind less well to (A/T)4sites containing TpA steps. We find that in (A/T)6tracts distamycin shows little discrimination between the various sites, binding approximately 2-fold stronger to TAATTA than (TA)3, T3A3and GAATTC. In contrast, Hoechst 33258 binds approximately 20-fold more tightly to GAATTC and TAATTA than T3A3and (TA)3. Hydroxyl radical footprinting reveals that both ligands bind in similar locations at the centre of each AT tract. At (A/T)10sites distamycin binds with similar affinity to T5A5, (TA)5and AATT, though bands in the centre of (TA)5are protected at approximately 50-fold lower concentration than those towards the edges. Hoechst 33258 shows a similar pattern of preference, with strong binding to AATT, T5A5and the centre of (TA)5. Hydroxyl radical footprinting reveals that at low concentrations both ligands bind at the centre of (TA)5and A5T5, while at higher concentrations ligand molecules bind to each end of the (A/T)10tracts. At T5A5two ligand molecules bind at either end of the site, even at the lowest ligand concentration, consistent with the suggestion that these compounds avoid the TpA step. Similar DNase I footprinting experiments with a DNA fragment containing T n (n = 3-6) tracts reveals that both ligands bind in the order T3< T4 << T5 = T6.     

RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication.

Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeIII fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L. Dailey, M. S. Caddle, N. Heintz, and N. H. Heintz, Mol. Cell. Biol. 10:6225-6235, 1990). Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)3-4 tracts, designated bend elements B1 to B5, that are spaced 10 bp apart. DNA bending directed by elements B1 to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4 degrees C. Bend element B5, which is in inverse orientation relative to elements B1 to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site. Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences. These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.