Exploring Hoogsteen and reversed-Hoogsteen duplex and triplex formation with tricyclo-DNA purine sequences

The duplex- and triplex-formation properties of the tricyclo-DNA purine decamer 5'p-gagaaggaaa-3' as a single strand or as part of a hairpin duplex with corresponding parallel and antiparallel pyrimidine DNA and RNA complements, as well as with antiparallel purine DNA and RNA complements, were investigated by UV melting curve analysis, circular dichroism spectroscopy, and gel mobility shift experiments. It was found that tricyclo-DNA forms very stable duplexes with the pyrimidine RNA and DNA complements not only in the Watson-Crick pairing mode, but also in the Hoogsteen one. Below pH 6.0, the tc-DNA/DNA and tc-DNA/RNA Hoogsteen duplexes were found to be more stable than the corresponding Watson-Crick DNA duplexes. Triplexes of the hairpin structure with parallel pyrimidine complements revealed even stronger Hoogsteen pairing relative to the duplexes, presumably due to structural preorganization phenomena. Triplex formation with antiparallel pyrimidine and purine third strands (reversed-Hoogsteen motif) could not be observed and seem to be unstable

VERIFICATION OF DNA PREDICTED PROTEIN SEQUENCES BY ENZYME HYDROLYSIS AND MASS SPECTROMETRIC ANALYSIS

The focus of this thesis lies in the development of a sensitive method for the analysis of protein primary structure which can be easily used to confirm the DNA sequence of a protein's gene and determine the modifications which are made after translation. This technique involves the use of dipeptidyl aminopeptidase (DAP) and dipeptidyl carboxypeptidase (DCP) to hydrolyze the protein and the mass spectrometric analysis of the dipeptide products.^ Dipeptidyl carboxypeptidase was purified from human lung tissue and characterized with respect to its proteolytic activity. The results showed that the enzyme has a relatively unrestricted specificity, making it useful for the analysis of the C-terminal of proteins. Most of the dipeptide products were identified using gas chromatography/mass spectrometry (GC/MS). In order to analyze the peptides not hydrolyzed by DCP and DAP, as well as the dipeptides not identified by GC/MS, a FAB ion source was installed on a quadrupole mass spectrometer and its performance evaluated with a variety of compounds.^ Using these techniques, the sequences of the N-terminal and C-terminal regions and seven fragments of bacteriophage P22 tail protein have been verified. All of the dipeptides identified in these analysis were in the same DNA reading frame, thus ruling out the possibility of a single base being inserted or deleted from the DNA sequence. The verification of small sequences throughout the protein sequence also indicates that no large portions of the protein have been removed after translation. ^

DNA incisions stimulate genetic instability of triplet repeat sequences related to human hereditary neurological diseases

The molecular mechanisms responsible for the expansion and deletion of trinucleotide repeat sequences (TRS) are the focus of our studies. Several hereditary neurological diseases including Huntington's disease, myotonic dystrophy, and fragile X syndrome are associated with the instability of TRS. Using the well defined and controllable model system of Escherichia coli, the influences of three types of DNA incisions on genetic instability of CTG•CAG repeats were studied: DNA double-strand breaks (DSB), single-strand nicks, and single-strand gaps. The DNA incisions were generated in pUC19 derivatives by in vitro cleavage with restriction endonucleases. The cleaved DNA was then transformed into E. coli parental and mutant strains. Double-strand breaks induced deletions throughout the TRS region in an orientation dependent manner relative to the origin of replication. The extent of instability was enhanced by the repeat length and sequence (CTG•CAG vs. CGG•CCG). Mutations in recA and recBC increased deletions, mutations in recF stabilized the TRS, whereas mutations in ruvA had no effect. DSB were repaired by intramolecular recombination, versus an intermolecular gene conversion or crossover mechanism. 30 nt gaps formed a distinct 30 nt deletion product, whereas single strand nicks and gaps of 15 nts did not induce expansions or deletions. Formation of this deletion product required the CTG•CAG repeats to be present in the single-stranded region and was stimulated by E. coli DNA ligase, but was not dependent upon the RecFOR pathway. Models are presented to explain the DSB induced instabilities and formation of the 30 nucleotide deletion product. In addition to the in vitro creation of DSBs, several attempts to generate this incision in vivo with the use of EcoR I restriction modification systems were conducted. ^

Non B-DNA structures and genomic instability associated with myotonic dystrophy type 2 (CCTG).(CAGG) repeats

There are many diseases associated with the expansion of DNA repeats in humans. Myotonic dystrophy type 2 is one of such diseases, characterized by expansions of a (CCTG)•(CAGG) repeat tract in intron 1 of zinc finger protein 9 (ZNF9) in chromosome 3q21.3. The DM2 repeat tract contains a flanking region 5' to the tract that consists of a polymorphic repetitive sequence (TG)14-25(TCTG)4-11(CCTG) n. The (CCTG)•(CAGG) repeat is typically 11-26 repeats in persons without the disease, but can expand up to 11,000 repeats in affected individuals, which is the largest expansion seen in DNA repeat diseases to date. This DNA tract remains one of the least characterized disease-associated DNA repeats, and mechanisms causing the repeat expansion in humans have yet to be elucidated. Alternative, non B-DNA structures formed by the expanded repeats are typical in DNA repeat expansion diseases. These sequences may promote instability of the repeat tracts. I determined that slipped strand structure formation occurs for (CCTG)•(CAGG) repeats at a length of 42 or more. In addition, Z-DNA structure forms in the flanking human sequence adjacent to the (CCTG)•(CAGG) repeat tract. I have also performed genetic assays in E. coli cells and results indicate that the (CCTG)•(CAGG) repeats are more similar to the highly unstable (CTG)•(CAG) repeat tracts seen in Huntington's disease and myotonic dystrophy type 1, than to those of the more stable (ATTCT)•(AGAAT) repeat tracts of spinocerebellar ataxia type 10. This instability, however, is RecA-independent in the (CCTG)•(CAGG) and (ATTCT)•(AGAAT) repeats, whereas the instability is RecA-dependent in the (CTG)•(CAG) repeats. Structural studies of the (CCTG)•(CAGG) repeat tract and the flanking sequence, as well as genetic selection assays may reveal the mechanisms responsible for the repeat instability in E. coli, and this may lead to a better understanding of the mechanisms contributing to the human disease state. ^

DNA alignment and Bayesian trees of partial sequences of COI, COIII and the haemocyanin functional unit f-g of 28 octopod species

Background: Octopods have successfully colonised the world's oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods. Results: Analysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence. Conclusions: This study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Induction of microsatellite instability by oxidative DNA damage

Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the β-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of β-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the β-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 106 wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency ≈300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N′-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This β-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.

Inactivation of DNA proofreading obviates the need for SOS induction in frameshift mutagenesis

Translesion synthesis at replication-blocking lesions requires the induction of proteins that are controlled by the SOS system in Escherichia coli. Of the proteins identified so far, UmuD′, UmuC, and RecA* were shown to facilitate replication across UV-light-induced lesions, yielding both error-free and mutagenic translesion-synthesis products. Similar to UV lesions, N-2-acetylaminofluorene (AAF), a chemical carcinogen that forms covalent adducts at the C8 position of guanine residues, is a strong replication-blocking lesion. Frameshift mutations are induced efficiently by AAF adducts when located within short repetitive sequences in a two-step mechanism; AAF adducts incorporate a cytosine across from the lesion and then form a primer-template misaligned intermediate that, upon elongation, yields frameshift mutations. Recently, we have shown that although elongation from the nonslipped intermediate depends on functional umuDC+ gene products, elongation from the slipped intermediate is umuDC+-independent but requires another, as yet biochemically uncharacterized, SOS function. We now show that in DNA Polymerase III-proofreading mutant strains (dnaQ49 and mutD5 strains), elongation from the slipped intermediate is highly efficient in the absence of SOS induction—in contrast to elongation from the nonslipped intermediate, which still requires UmuDC functions.

Short DNA Fragments without Sequence Similarity Are Initiation Sites for Replication in the Chromosome of the Yeast Yarrowia lipolytica

We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (Vernis et al., 1997). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowia origins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity.

Differentiation of Chromatin during DNA Elimination in Euplotes crassus

In Euplotes crassus, most of the micronuclear genome is eliminated during formation of a transcriptionally active macronucleus. To understand how this is mediated throughout the genome, we have examined the chromatin structure of the macronucleus-destined sequences and Tec transposons, which are dispersed in 15,000 copies in the micronuclear genome and completely eliminated during formation of the macronuclear genome. Whereas the macronucleus-destined sequences show a typical pattern of nucleosomal repeats in micrococcal nuclease digests, the Tec element chromatin structure digests to a nucleosome-like repeat pattern that is not typical: the minimum digestion products are ∼300–600 base pairs, or “subnucleosomal,” in size. In addition, the excised, circular forms of the Tec elements are exceedingly resistant to nucleases. Nevertheless, an underlying nucleosomal structure of the Tec elements can be demonstrated from the size differences between repeats in partial micrococcal nuclease digests and by trypsin treatment of nuclei, which results in mononucleosome-sized products. Characterization of the most micrococcal nuclease–resistant DNA indicates that micronuclear telomeres are organized into a chromatin structure with digestion properties identical to those of the Tec elements in the developing macronucleus. Thus, these major repetitive sequence components of the micronuclear genome differ in their chromatin structure from the macronuclear-destined sequences during DNA elimination. The potential role of developmental stage–specific histone variants in this chromatin differentiation is discussed.

Multiple independent transpositions of mitochondrial DNA control region sequences to the nucleus

Transpositions of mtDNA sequences to the nuclear genome have been documented in a wide variety of individual taxa, but little is known about their taxonomic frequency or patterns of variation. We provide evidence of nuclear sequences homologous to the mtDNA control region in seven species of diving ducks (tribe Aythyini). Phylogenetic analysis places each nuclear sequence as a close relative of the mtDNA haplotypes of the specie(s) in which it occurs, indicating that they derive from six independent transposition events, all occurring within the last ≈1.5 million years. Relative-rate tests and comparison of intraspecific variation in nuclear and mtDNA sequences confirm the expectation of a greatly reduced rate of evolution in the nuclear copies. By representing mtDNA haplotypes from ancestral populations, nuclear insertions may be valuable in some phylogenetic analyses, but they also confound the accurate determination of mtDNA sequences. In particular, our data suggest that the presumably nonfunctional but more slowly evolving nuclear sequences often will not be identifiable by changes incompatible with function and may be preferentially amplified by PCR primers based on mtDNA sequences from related taxa.

Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47–81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

The Homeodomain Resource: sequences, structures, DNA binding sites and genomic information

The Homeodomain Resource is an annotated collection of non-redundant protein sequences, three-dimensional structures and genomic information for the homeodomain protein family. Release 3.0 contains 795 full-length homeodomain-containing sequences, 32 experimentally-derived structures and 143 homeo­box loci implicated in human genetic disorders. Entries are fully hyperlinked to facilitate easy retrieval of the original records from source databases. A simple search engine with a graphical user interface is provided to query the component databases and assemble customized data sets. A new feature for this release is the addition of DNA recognition sites for all human homeodomain proteins described in the literature. The Homeodomain Resource is freely available through the World Wide Web at http://genome.nhgri.nih.gov/homeodomain.

Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery

The product of the herpes simplex virus type 1 UL28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of UL28 in this process, purified UL28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for UL28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by UL28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by UL28 protein. To determine the relevance of the observed UL28 protein–pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of UL28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by UL28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the UL28 protein–pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the UL28-encoded component of the herpesvirus cleavage and packaging machinery.