Herpesvirus ateles DNA and Its Homology with Herpesvirus saimiri Nucleic Acid

Analysis of the structural organization of Herpesvirus ateles DNA shows that two types of viral DNA molecules are encapsidated in virions: (i) M-genomes, which contain 74% light sequences (L-DNA, 38% guanine plus cytosine) and 26% highly repetitive heavy sequences (H-DNA, 75% guanine plus cytosine), and (ii) defective H-genomes, which consist exclusively of repetitive H-DNA. The structure of M-genomes from H. ateles consists of an L-DNA region of about 70 x 10(6) daltons inserted between H-DNA termini of variable length. M-genomes with a shorter H-DNA region at one end of the molecule have a long stretch of H-DNA at the other end, resulting in a total molecular weight of 89.8 +/- 8.5 x 10(6). Thus it resembles the structure of M-genomes of H. saimiri. H-DNA of the two independent H. ateles isolates, strains 810 and 73, reveals different patterns after cleavage with restriction endonuclease Sma I. H-DNA of H. ateles 810 appears to consist of identical tandem repeat units with a molecular weight of 1,035,000; the H-DNA repeat unit of strain 73 is shorter (930,000 molecular weight). Corresponding DNA sequences of the two H. ateles strains (810 and 73) are completely homologous in cross-hybridizations. However, a discrete nucleotide sequence divergence between these virus strains is detected by measuring melting temperatures (T(m)) of DNA hybrid molecules. Some homology exists between H. ateles and H. saimiri DNA. Hybridization of L-DNA from H. ateles with L-DNA from H. saimiri shows about a 35% homology between the respective L-DNA sequences; the resulting heteroduplex molecules show a decrease of T(m) by 13.5 degrees C, corresponding to about a 9% mismatching in cross-hybridizing parts of L-regions. Very little homology is found between H-DNA of H. ateles and H. saimiri.     

DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells

Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA.     

H-DNA and Z-DNA in the mouse c-Ki-ras promoter.

The mouse c-Ki-ras protooncogene promoter contains a homopurine-homopyrimidine domain that exhibits S1 nuclease sensitivity in vitro. We have studied the structure of this DNA region in a supercoiled state using a number of chemical probes for non-B DNA conformations including diethyl pyrocarbonate, osmium tetroxide, chloroacetaldehyde, and dimethyl sulfate. The results demonstrate that two types of unusual DNA structures formed under different environmental conditions. A 27-bp homopurine-homopyrimidine mirror repeat adopts a triple-helical H-DNA conformation under mildly acidic conditions. This H-DNA seems to account for the S1 hypersensitivity of the promoter in vitro, since the observed pattern of S1 hypersensitivity at a single base level fits well with the H-DNA formation. Under conditions of neutral pH we have detected Z-DNA created by a (CG)5-stretch, located adjacent to the homopurine-homopyrimidine mirror repeat. The ability of the promoter DNA segment to form non-B structures has implications for models of gene regulation.     

The structure of intramolecular triplex DNA: atomic force microscopy study

We applied atomic force microscopy (AFM) for direct imaging of intramolecular triplexes (H-DNA) formed by mirror-repeated purine-pyrimidine repeats and stabilized by negative DNA supercoiling. H-DNA appears in atomic force microscopy images as a clear protrusion with a different thickness than DNA duplex. Consistent with the existing models, H-DNA formation results in a kink in the double helix path. The kink forms an acute angle so that the flanking DNA regions are brought in close proximity. The mobility of flanking DNA arms is limited compared with that for cruciforms and three-way junctions. Structural properties of H-DNA may be important for promoter-enhancer interactions and other DNA transactions.     

Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction

The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19. Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells. A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.     

Nucleosome positioning signals and potential H-DNA within the DNA sequence of the imprinting control region of the mouse Igf2r gene

The imprinting control region within the second intron of the mouse Igf2r gene contains a CpG island comprising direct repeats, an imprinting box and the Air antisense promoter which is blocked by the methylation imprint on the active maternal allele. We have investigated the structural features of this DNA, including a mapping of all nucleosome positioning signals within the nucleotide sequence. A discrete series of strong positioning signals distinguished the direct repeat region from the much more diverse positioning capacity of the sequence encompassing the known regulatory elements. At only a few locations did CpG methylation modulate the use of this positioning information. Direct effects upon histone-DNA interactions are therefore unlikely to contribute significantly to the means by which the imprint may establish allele-specific chromatin architecture and determine Air expression. A strand-specific obstruction to DNA polymerase was observed between the repeat and regulatory regions. The same region adopts triple-stranded H-DNA structures in supercoiled DNA, according to pH and divalent cation exposure. Methylation did not modulate the occurrence or form of this structure under the conditions tested. This finding nevertheless adds to the repertoire of potential H-DNA structures found in the vicinity of regulatory sequences-here, in an imprinting context.     

Structure of nonintegrated, circular Herpesvirus saimiri and Herpesvirus ateles genomes in tumor cell lines and in vitro-transformed cells.

Nonintegrated, circular DNA molecules of Herpesvirus saimiri and Herpesvirus ateles were found in five lymphoid cell lines originating from tumor tissues or established by in vitro immortalization of T lymphocytes. The arrangement of unique (L) and repetitive (H) DNA sequences in circular viral genomes was analyzed by partial denaturation mapping followed by visualization with an electron microscope. Three types of circular viral DNA structures were found. (i) The virus-producing cell line RLC, which is derived from an H. ateles-induced rabbit lymphoma, contains circular viral genomes which consist of a single L-DNA and a single H-DNA region, both the same length as in virion DNA. (ii) The circular viral genomes of the nonproducer cell lines H1591 and A1601, in vitro transformed by H. saimiri and H. ateles, respectively, have deletions in the unique L-DNA region and larger H-DNA regions. Cell line A1601 lacks about 8% of virion L-DNA, and H1591 cells lack about 40% of viral L-DNA information. (iii) The nonproducing H. saimiri tumor cell lines 1670 and 70N2 harbor viral genomes with two L-DNA and two H-DNA regions, respectively. Both types of circular molecules have a long and a short L-segment. The sequence arrangements of circular DNA molecules from H. saimiri-transformed cell lines were compared with those of linear virion DNA by computer alignment of partial denaturation histograms. The L-DNA deletion in cell line H1591 was found to map in the right half of the virion DNA. Comparison of the denaturation patterns of both L regions of cell lines 1670 and 70N2 identified the short L regions as subsets of the long L regions. Thus, circular viral DNA molecules of all four nonproducer cell lines represent defective genomes.     

Suppression of cyclobutane and mean value of 6-4 dipyrimidines formation in triple-stranded H-DNA

We have determined the effect of H-DNA formation on the distributions of two ultraviolet (UV) light induced photoproducts--cyclobutane dipyrimidines and mean value of 6-4 dipyrimidines. A region of DNA containing the sequence (dT-dC)18.(dA-dG)18 was treated under conditions that specifically yield the triple-stranded H-y3 or H-y5 DNA structure and then irradiated with UV. The positions of cyclobutane dipyrimidines and mean value of 6-4 dipyrimidines were determined by T4 endonuclease V cleavage and by hot piperidine cleavage, respectively. Formation of H-DNA structures greatly decreased the photoproduct yield in the (dT-dC)18.(dA-dG)18 region but not elsewhere in the DNA. Suppression of photoproduct formation is greater in half of the repeat, reflecting whether the DNA is in the H-y3 or H-y5 conformation. Within the repeat, the suppression was less in the middle and toward the ends. Models for the suppression of photoproduct formation in H-DNA and the possible utility of our findings are discussed.     

Chemical determinants of DNA bending at adenine-thymine tracts

DNA fragments having homopolymeric adenine-thymine tracts phased with the helix screw are known to be bent. According to our working model, adenine-thymine tracts adopt a polymorphic structure (H-DNA), and juxtaposition of H-DNA with B-DNA results in bending at the junction between the two structures. We incorporated different base analogues in addition to the four ordinary bases into oligonucleotides; ligated multimers of oligonucleotide duplexes were run on polyacrylamide gels. By comparison of gel mobility data for different sequences, we identified factors both necessary and irrelevant for bending, corresponding to the formation of H-DNA. The 5-methyl group on pyrimidines is not essential, and the 2-amino group on purines interferes with the formation of H-DNA, either because it provides a third H bond between the bases or because it alters water structure in the minor groove. The strong base stacking of A may be an important contributing factor to stabilization of the anomalous DNA structure responsible for bending.     

Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA.

We have used computer-assisted methods to search large amounts of the human, yeast and Escherichia coli genomes for inverted repeat (IR) and mirror repeat (MR) DNA sequence patterns. In highly supercoiled DNA some IRs can form cruciforms, while some MRs can form intramolecular triplexes, or H-DNA. We find that total IR and MR sequences are highly enriched in both eukaryotic genomes. In E. coli, however, only total IRs are enriched, while total MRs only occur as frequently as in random sequence DNA. We then used a set of experimentally derived criteria to predict which of the total IRs and MRs are most likely to form cruciforms or H-DNA in supercoiled DNA. We show that strong cruciform forming sequences occur at a relatively high frequency in yeast (1/19 700 bp) and humans (1/41 800 bp), but that H-DNA forming sequences are abundant only in humans (1/49 400 bp). Strong cruciform and H-DNA forming sequences are not abundant in the E.coli genome. These results suggest that cruciforms and H-DNA may have a functional role in eukaryotes, but probably not prokaryotes.     

The effect of zinc on the secondary structure of d(GA.TC)n DNA sequences of different length: a model for the formation *H-DNA.

Alternating d(GA.TC)n DNA sequences are known to undergo transition to *H-DNA in the presence of zinc. Here, the effect of zinc on the secondary DNA structure of d(GA.TC)n sequences of different length (n = 5, 8, 10 and 19) was determined. Short d(GA.TC)n sequences form *H-DNA with a higher difficulty than longer ones. At bacterial negative superhelical density (- sigma = 0.05), zinc still induces transition to the *H-DNA conformation at a d(GA.TC)10 sequence but shorter sequences do not form *H-DNA. Transition to *H-DNA at a d(GA.TC)8 sequence is observed under conditions which destabilize the DNA double helix such as high negative supercoiling or low ionic strength. Our results indicate that a first step in the transition to *H-DNA is the formation of a denaturation bubble at the centre of the repeated DNA sequence, suggesting that the primary role of zinc is to induce a local denaturation of the DNA double helix. Subsequently, zinc might also participate in the stabilization of the altered DNA conformation through its direct interaction with the bases. Based on these results a model for the formation of *H-DNA is proposed.