Craig J. Benham

OriDB: a DNA replication origin database

Replication of eukaryotic chromosomes initiates at multiple sites called replication origins. Replication origins are best understood in the budding yeast Saccharomyces cerevisiae, where several complementary studies have mapped their locations genome-wide. We have collated these datasets, taking account of the resolution of each study, to generate a single list of distinct origin sites. OriDB provides a web-based catalogue of these confirmed and predicted S.cerevisiae DNA replication origin sites. Each proposed or confirmed origin site appears as a record in OriDB, with each record comprising seven pages. These pages provide, in text and graphical formats, the following information: genomic location and chromosome context of the origin site; time of origin replication; DNA sequence of proposed or experimentally confirmed origin elements; free energy required to open the DNA duplex (stress-induced DNA duplex destabilization or SIDD); and phylogenetic conservation of sequence elements. In addition, OriDB encourages community submission of additional information for each origin site through a User Notes facility. Origin sites are linked to several external resources, including the Saccharomyces Genome Database (SGD) and relevant publications at PubMed. Finally, a Chromosome Viewer utility allows users to interactively generate graphical representations of DNA replication data genome-wide. OriDB is available at .

Loss of FBP function arrests cellular proliferation and extinguishes c‐ myc expression

The c‐myc regulatory region includes binding sites for a large set of transcription factors. The present studies demonstrate that in the absence of FBP [far upstream element (FUSE)‐binding protein], which binds to the single‐stranded FUSE, the remainder of the set fails to sustain endogenous c‐myc expression. A dominant‐negative FBP DNA‐binding domain lacking effector activity or an antisense FBP RNA, expressed via replication‐defective adenovirus vectors, arrested cellular proliferation and extinguished native c‐myc transcription from the P1 and P2 promoters. The dominant‐negative FBP initially augmented the single‐stranded character of FUSE; however, once c‐myc expression was abolished, melting at FUSE could no longer be supported. In contrast, with antisense FBP RNA, the single‐stranded character of FUSE decreased monotonically as the transcription of endogenous c‐myc declined. Because transcription is the major source of super‐coiling in vivo, we propose that by binding torsionally strained DNA, FBP measures promoter activity directly. We also show that FUSE is predicted to behave as a torsion‐regulated switch poised to regulate c‐myc and to confer a higher order regulation on a large repertoire of factors.

Activation of Gene Expression by a Novel DNA Structural Transmission Mechanism That Requires Supercoiling-induced DNA Duplex Destabilization in an Upstream Activating Sequence

We have previously demonstrated that integration host factor (IHF)-mediated activation of transcription from theilvPG promoter of Escherichia colirequires a supercoiled DNA template and occurs in the absence of specific interactions between IHF and RNA polymerase. In this report, we describe a novel, supercoiling-dependent, DNA structural transmission mechanism for this activation. We provide theoretical evidence for a supercoiling-induced DNA duplex destabilized (SIDD) structure in the A + T-rich, ilvPG regulatory region between base pair positions +1 and −160. We show that the region of this SIDD sequence immediately upstream of an IHF binding site centered at base pair position −92 is, in fact, destabilized by superhelical stress and that this duplex destabilization is inhibited by IHF binding. Thus, in the presence of IHF, the negative superhelical twist normally absorbed by this DNA structure in the promoter distal half of the SIDD sequence is transferred to the downstream portion of the SIDD sequence containing the ilvPG promoter site. This IHF-mediated translocation of superhelical energy facilitates duplex destabilization in the −10 region of the downstream ilvPG promoter and activates transcription by increasing the rate of open complex formation.

Energetics of the strand separation transition in superhelical DNA

In this paper the values of three free energy parameters governing the superhelical strand separation transition are determined by analysis of available experimental data. These are the free energy, a, needed to initiate a run of separation, the torsional stiffness, C, associated with interstrand winding of the two single strands comprising a separated site and the coefficient, K, of the quadratic free energy associated to residual linking. The experimental data used in this analysis are the locations and relative amounts of strand separation occurring in the pBR322 DNA molecule and the measured residual linking, both evaluated over a range of negative linking differences. The analytic method used treats strand separation as a heteropolymeric, co-operative, two-state transition to a torsionally deformable alternative conformation, which takes place in a circular DNA molecule constrained by the constancy of its linking number. The values determined for these parameters under the experimental conditions (T = 310 K, pH = 7.0, monovalent cation concentration = 0.01 M) are a = 10.84(+/- 0.2) kcal/mol, C = 2.5(+/- 0.3) x 10(-13) erg/rad2 and K = 2350(+/- 80) RT/N, where N is the molecular length in base-pairs. In order to assess the accuracy of the authors theoretical methods, these free energy parameters are incorporated into the analysis of superhelical strand separation in different molecules and under other conditions than those used in their evaluation. First, the temperature dependence of transition is treated, then superhelical strand separation is analyzed in a series of DNA molecules having systematic sequence modifications, and the results of these theoretical analyses are compared with those from experiments. In all molecules, transition is predicted in the range of linking differences where it is seen experimentally. Moreover, it occurs at the specific sequence locations that the analysis predicts, and with approximately the predicted relative amounts of transition at each location. The known sensitivities of this transition to changes of temperature and to small sequence modifications are predicted in a quantitatively precise manner by the theoretical results. The demonstrated high-level precision of these theoretical methods provides a tool for the screening of DNA sequences for sites susceptible to superhelical strand separation, some of which may have regulatory or other biological significance.

Fatal connections: When DNA ends meet on the nuclear matrix

A damaged nucleus has long been regarded simply as a “bag of broken chromosomes,” with the DNA free ends moving around and forming connections with randomly encountered partners. Recent evidence shows this picture to be fundamentally wrong. Chromosomes occupy specific nuclear domains within which only limited movement is possible. In a human diploid nucleus, 6.6 × 109 base pairs (bp) of DNA are compartmentalized into chromosomes in a way that allows stringent control of replication, differential gene expression, recombination and repair. Most of the chromatin is further organized into looped domains by the dynamic binding of tethered bases to a network of intranuclear proteins, the so‐called nuclear scaffold or matrix. Thus, DNA movement is severely curtailed, which limits the number of sites where interchanges can occur. This intricate organizational arrangement may render the genome vulnerable to processes that interfere with DNA repair. Both lower and higher eukaryotic cells perform homologous recombination (HR) and illegitimate recombination (IR) as part of their survival strategies. The repair processes comprising IR must be understood in the context of DNA structural organization, which is fundamentally different in prokaryotic and eukaryotic genomes. In this paper we first review important cellular processes including recombination, DNA repair, and apoptosis, and describe the central elements involved. Then we review the different DNA targets of recombination, and present recent evidence implicating the nuclear matrix in processes which can induce either repair, translocation, deletion, or apoptosis. J. Cell. Biochem. Suppl. 35:3–22, 2000.

Integration site choice of a feline immunodeficiency virus vector.

ABSTRACT We mapped 226 unique integration sites in human hepatoma cells following gene transfer with a feline immunodeficiency virus (FIV)-based lentivirus vector. FIV integrated across the entire length of the transcriptional units. Microarray data indicated that FIV integration favored actively transcribed genes. Approximately 21% of FIV integrations within transcriptional units occurred in genes regulated by the LEDGF/p75 transcriptional coactivator. DNA in regions of FIV insertion sites exhibited a “bendable” structure and a pattern of duplex destabilization favoring strand separation. FIV integration preferences are more similar to those of primate lentiviruses and distinct from those of Moloney murine leukemia virus, avian sarcoma leukosis virus, and foamy virus.

Promoter prediction and annotation of microbial genomes based on DNA sequence and structural responses to superhelical stress

BackgroundIn our previous studies, we found that the sites in prokaryotic genomes which are most susceptible to duplex destabilization under the negative superhelical stresses that occur in vivo are statistically highly significantly associated with intergenic regions that are known or inferred to contain promoters. In this report we investigate how this structural property, either alone or together with other structural and sequence attributes, may be used to search prokaryotic genomes for promoters.ResultsWe show that the propensity for stress-induced DNA duplex destabilization (SIDD) is closely associated with specific promoter regions. The extent of destabilization in promoter-containing regions is found to be bimodally distributed. When compared with DNA curvature, deformability, thermostability or sequence motif scores within the -10 region, SIDD is found to be the most informative DNA property regarding promoter locations in the E. coli K12 genome. SIDD properties alone perform better at detecting promoter regions than other programs trained on this genome. Because this approach has a very low false positive rate, it can be used to predict with high confidence the subset of promoters that are strongly destabilized. When SIDD properties are combined with -10 motif scores in a linear classification function, they predict promoter regions with better than 80% accuracy. When these methods were tested with promoter and non-promoter sequences from Bacillus subtilis, they achieved similar or higher accuracies. We also present a strictly SIDD-based predictor for annotating promoter sequences in complete microbial genomes.ConclusionIn this report we show that the propensity to undergo stress-induced duplex destabilization (SIDD) is a distinctive structural attribute of many prokaryotic promoter sequences. We have developed methods to identify promoter sequences in prokaryotic genomes that use SIDD either as a sole predictor or in combination with other DNA structural and sequence properties. Although these methods cannot predict all the promoter-containing regions in a genome, they do find large sets of potential regions that have high probabilities of being true positives. This approach could be especially valuable for annotating those genomes about which there is limited experimental data.

The analysis of stress-induced duplex destabilization in long genomic DNA sequences.

We present a method for calculating predicted locations and extents of stress-induced DNA duplex destabilization (SIDD) as functions of base sequence and stress level in long DNA molecules. The base pair denaturation energies are assigned individually, so the influences of near neighbors, methylated bases, adducts, or lesions can be included. Sample calculations indicate that copolymeric energetics give results that are close to those derived when full near-neighbor energetics are used; small but potentially informative differences occur only in the calculated SIDD properties of moderately destabilized regions. The method presented here for analyzing long sequences calculates the destabilization properties within windows of fixed length N, with successive windows displaced by an offset distance d(o). The final values of the relevant destabilization parameters for each base pair are calculated as weighted averages of the values computed for each window in which that base pair appears. This approach implicitly assumes that the strength of the direct coupling between remote base pairs that is induced by the imposed stress attenuates with their separation distance. This strategy enables calculations of the destabilization properties of DNA sequences of any length, up to and including complete chromosomes. We illustrate its utility by calculating the destabilization properties of the entire E. coli genomic DNA sequence. A preliminary analysis of the results shows that promoters are associated with SIDD regions in a highly statistically significant manner, suggesting that SIDD attributes may prove useful in the computational prediction of promoter locations in prokaryotes.

Activation of transcription initiation from a stable RNA promoter by a Fis protein‐mediated DNA structural transmission mechanism

The leuV operon of Escherichia coli encodes three of the four genes for the tRNA1Leu isoacceptors. Transcription from this and other stable RNA promoters is known to be affected by a cis‐acting UP element and by Fis protein interactions with the carboxyl‐terminal domain of the α‐subunits of RNA polymerase. In this report, we suggest that transcription from the leuV promoter also is activated by a Fis‐mediated, DNA supercoiling‐dependent mechanism similar to the IHF‐mediated mechanism described previously for the ilvPG promoter (S. D. Sheridan et al., 1998, J Biol Chem 273: 21298–21308). We present evidence that Fis binding results in the translocation of superhelical energy from the promoter‐distal portion of a supercoiling‐induced DNA duplex destabilized (SIDD) region to the promoter‐proximal portion of the leuV promoter that is unwound within the open complex. A mutant Fis protein, which is defective in contacting the carboxyl‐terminal domain of the α‐subunits of RNA polymerase, remains competent for stimulating open complex formation, suggesting that this DNA supercoiling‐dependent component of Fis‐mediated activation occurs in the absence of specific protein interactions between Fis and RNA polymerase. Fis‐mediated translocation of superhelical energy from upstream binding sites to the promoter region may be a general feature of Fis‐mediated activation of transcription at stable RNA promoters, which often contain A+T‐rich upstream sequences.

Theoretical analysis of competitive conformational transitions in torsionally stressed DNA

Abstract This paper presents a theoretical analysis of the conformation of a torsionally deformed segment of DNA containing two sites susceptible to stress-induced transitions in secondary structure. A mechanical analysis of the ensuing competitive behavior is developed and applied to several phenomena of possible biological relevance. First, a molecular lesion which disrupts base pairing without strand breakage (such as a pyrimidine dimer) is shown to provide an effective nucleation site for further stress-induced denaturation. A competition is established between strand separation at this lesion site and at the A + T-richest portion of the molecule. The relative importance of these two forms of melting is shown to depend upon the A + T-content of the sites involved, segment length, local environmental conditions and the magnitude of the imposed torsional deformation. A possible alternative mode of behavior of a stressed segment of DNA involves transitions from B-form to Z-form. The second application of this theory analyzes the interplay between B → Z transitions and local denaturation in torsionally stressed DNA. Finally, local melting is shown to be energetically preferred over transitions to A-form under physiologically reasonable conditions in vitro , due primarily to the greater degree of unwinding involved in melting. The mechanical theory presented here makes several simplifying assumptions regarding the nature of the transitions and the sequences involved. First, the theory is developed explicitly for the competition between two sites of possible transition, with no further consideration given to conformational degeneracy or sequence effects. These sites are regarded as being uniform in composition. A multistate, heteropolymeric statistical mechanical transition theory is required to account rigorously for degeneracy and the influence of base sequence. A preliminary formulation of such a theory is used to analyze the denaturation of a segment containing a site of disrupted base pairing.