In vivo assessment of the Z-DNA-forming potential of d(CA.GT)n and d(CG.GC)n sequences cloned into SV40 minichromosomes

Alternating repeated d(CA.GT)n and d(CG.GC)n sequences constitute a significant proportion of the simple repeating elements found in eukaryotic genomic DNA. These sequences are known to form left-handed Z-DNA in vitro. In this paper, we have addressed the question of the in vivo determination of the Z-DNA-forming potential of such sequences in eukaryotic chromatin. For this purpose, we have investigated the ability of a d(CA.GT)30 sequence and a d(CG.GC)5 sequence to form left-handed Z-DNA when cloned into simian virus 40 (SV40) minichromosomes at two different positions: the TaqI site, which occurs in the intron of the T-antigen gene, and the HpaII site, which is located in the late promoter region within the SV40 control region. Formation of Z-DNA at the inserted repeated sequences was analyzed through the change in DNA linkage associated with the B to Z transition. Our results indicate that regardless of: (1) the site of insertion (either TaqI or HpaII), (2) the precise moment of the viral lytic cycle (from 12 h to 48 h postinfection) and (3) the condition of incorporation of the SV40 recombinants to the host cells (either as minichromosomes or as naked DNA, relaxed or negatively supercoiled), neither the d(CA.GT)30 nor the d(CG.GC)5 sequence are stable in the left-handed Z-DNA conformation in the SV40 minichromosome. The biological relevance of these results is discussed.     

Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.     

Construction of a viable simian virus 40 variant that carries a poly[d(GT) . d(CA)] insertion.

A 90-base-pair tract of a simple sequence composed of alternating guanosine and thymidine nucleotide residues (poly[d(GT) . d(CA)]) was inserted into the simian virus 40 genome at nucleotide 2666 (0.17 map units). The poly[d(GT) . d(CA)] insertion was stably maintained in the viral genome, but the variant virus grew more slowly than simian virus 40.     

The effect of the simple repeating d(CG.GC)n, d(CA.GT)n, and d(A.T)n DNA sequences on the nucleosomal organization of SV40 minichromosomes.

The effect of several simple repeating DNA sequences--d(CG.GC)5, d(CA.GT)30, and d(A.T)60--on the nucleosomal organization of the SV40 minichromosome is analyzed. These three different sequences were cloned at the Hpa II site of SV40 (position 346) which occurs at the 3' border of the nucleosome-free SV40 control region. Our results show that neither the d(A.T)60 sequence nor the d(CG.GC)5 sequence appear to have any relevant effect on the nucleosomal organization of the region of the minichromosome surrounding the inserted repeated sequence. Both sequences are hypersensitive to micrococcal nuclease cleavage in the minichromosome, indicating that they are not organized into nucleosomes. On the other hand, the d(CA.GT)30 sequence is found organized as nucleosomes and causes the delocation of nucleosomes in the minichromosomal region close to the inserted repeated sequence.     

RecA independent recombination of poly[d(GT)-d(CA)] in pBR322.

Short sequence tracts composed of alternating guanosine and thymidine nucleotide residues poly[d(GT)-d(CA)] carried in a derivative of pBR322 were recombinogenic in a recA host. Recombination brought about by poly[d(GT)-d(CA)] tracts displayed two interesting properties: (i) the reaction was quasi-sequence-specific in that while recombination usually occurred between two poly[d(GT)-d(CA)] tracts, recombination also occurred between sequences bordering the dinucleotide repeats. (ii) recombination was enhanced when two poly[d(GT)-d(CA)] tracts were clustered within 250 base pairs of each other, but not when the repeats were separated by 3 kilobase pairs. The mechanism by which poly[d(GT)-d(CA)] stimulated recombination remains to be determined, but the behavior of these sequences is consistent with the idea that general recombination in E. coli may involve formation of Z-DNA.     

A study of the B-Z transition of the AC-rich region of the repeat unit of a satellite DNA from Cebus by means of chemical probes

The conformational changes induced by negative supercoiling in the AC-rich region of the repeat unit of a Cebus satellite DNA has been studied by chemical probes sensitive to alterations in DNA conformation. This region is constituted of a (GT/CA)n stretch (15 less than or equal to n less than or equal to 18) associated to a sequence rich in GT/CA. At high superhelical density, at least 100 base pairs in the AC-rich region adopt the Z conformation as judged by diethyl pyrocarbonate reactivity. This is confirmed by diethyl pyrocarbonate footprinting of the complex between antibodies to Z-DNA and the AC-rich region. Osmium tetroxide and hydroxylamine reveal some distortions of the Z double helix in the (GT/CA)n stretch also. The terminal T residues of the stretch are hyperreactive with osmium tetroxide; the terminal left C residues but not the terminal right C residues are hyperreactive with hydroxylamine. Substitution of a few base pairs in the middle of the (GT/CA)n stretch induces also some distortions of the Z double helix. In the GT/CA-rich sequence, distortion of the Z double helix is also supported by the hyperreactivity of osmium tetroxide with several T and C residues.     

Genomic DNA sequence for human C-reactive protein

The gene for the prototype acute phase reactant, C-reactive protein, has been isolated from two lambda phage libraries containing inserted human DNA fragments using synthetic oligonucleotide probes. Nucleotide sequence analysis indicates that after coding for a signal peptide of 18 amino acids and the first two amino acids of the mature protein, there is an intron of 278 base pairs followed by the nucleotide sequence for the remaining 204 amino acids. The intron is unusual in that it contains on the positive strand a poly(A) stretch 16 nucleotides long and a poly(GT) region 30 nucleotides long which could adopt the Z-form of DNA. The nucleotide sequence reported here confirms the amino acid sequence of mature C-reactive protein as originally reported except that it codes for an additional 19 amino acids beginning at position 62. Thus DNA sequence analysis predicts that the mature protein consists of 206 amino acids rather than 187 as originally reported. The mRNA cap site is located 104 nucleotides from the start of the signal peptide and there is a 3' noncoding region 1.2 kilobase pairs in length. The gene has a typical promoter containing the sequences TATAAAT and CAAT 29 and 81 base pairs upstream, respectively, of the cap site.     

Locations and contexts of sequences that hybridize to poly(dG-dT).(dC-dA) in mammalian ribosomal DNAs and two X-linked genes.

Sequences located several kilobases both 5' and 3' of the stably transcribed portion of several genes hybridize to radio-labeled pure fragments of the alternating sequence poly (dG-dT) (dC-dA) ["poly(GT)"]. The genes include the ribosomal DNA of mouse, rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300 bp of adjoining DNA show perfect runs of poly(GT) (greater than 30bp) in all but the human 5' region of rDNA, which shows a somewhat different alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of the locations of tracts of alternating purine:pyrimidine between evolutionarily diverse species is consistent with a possible functional role for these sequences.     

Determination of the DNA conformation of the simian virus 40 (SV40) enhancer in SV40 minichromosomes

The simian virus 40 (SV40) enhancer contains three 8-bp purine-pyrimidine alternating sequences which are known to adopt the left-handed Z-DNA conformation in vitro. In this paper, we have undertaken the determination of the DNA conformation adopted by these Z-motifs in the SV40 minichromosome. We have analyzed the presence of Z-DNA through the change in linkage which should accompany formation of this left-handed conformation. Our results indicate that, regardless of the precise moment of the viral lytic cycle at which minichromosomes are harvested and the condition of the transfected DNA, either relaxed or negatively supercoiled, none of the three Z motifs of the SV40 enhancer exist to a significant extent as Z-DNA in SV40 minichromosomes. The SV40 enhancer adopts predominantly a right-handed B-DNA conformation in vivo.     

Interruptions of (CG)n sequences by GG, TG and CA need not prevent B to Z transition in solution.

Oligonucleotides containing alternating purines-pyrimidines with AT base pairs have been shown to exist in the Z-form preferably in solid state. We report that oligodeoxyribonucleotides with GG, TG and CA interruptions in their alternating CG sequences can undergo B to Z transition in solution in the absence of any chemical modification or topological constraint. The sequences, d(CGCGCGGCGCGC) and d(CGTGCGCACG) have been synthesised and shown to adopt Z- conformation in presence of millimolar concentrations of Ni2+ under low water activity conditions. Significance of GG, TG and CA interruptions in the B to Z transition is discussed.     

Assay of centromere function using a human artificial chromosome

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the α21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1–5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the α21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that α21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes. Received: 22 August 1998 / Accepted: 28 August 1998     

UV-induced interstrand cross-linking of d(GT)n.d(CA)n is facilitated by a structural transition

Photochemical alterations following ultraviolet irradiation of the alternating copolymer d(GT)n.d(CA)n were studied. We found that in solution conditions which produced circular dichroism spectra compatible with B-form or A-form DNA, no interstrand cross-linking or photoproduct formation could be demonstrated. Zimmer et al. (Zimmer, C., Tymen, S., Marck, C., and Guschlbaumer, W. (1982) Nucleic Acids Res. 10, 1081-1091) and Vorlickova et al. (Vorlickova, M., Kypr, J., Sotkrova, S., Sponar, J. (1982) Nucleic Acids Res. 10, 1071-1080) have reported a number of solution conditions which produce a structural transition of this polymer characterized by a negative deviation of the circular dichroism spectrum in the region of 280 nm. The nature of this transition has not yet been elucidated. Following ultraviolet irradiation of d(GT)n.d(CA)n under two conditions which produce this transition (manganese solution or ethanol plus trace salts solution) we found ultraviolet dose-dependent interstrand cross-linking as well as dose-dependent formation of thymine-containing photoproduct. Interstrand cross-linking is demonstrated by two criteria: increase in polymer size as detected by alkaline agarose gel electrophoresis, and generation of intermediate density material in alkaline cesium sulfate isopycnic gradients. The thymine-containing photo-product was demonstrated by thin layer chromatography of acid hydrolysates of the polymer. The photo-product is at least partially photoreversible. These findings suggest that the geometry of the alternative conformation is such that pyrimidines from different strands are closely approximated, allowing for photodimerization.     

Divalent cations stimulate preferential recognition of a viral DNA end by HIV-1 integrase.

In the presence of a divalent metal cofactor (Mg2+ or Mn2+), retroviral-encoded integrase (IN) catalyzes two distinct reactions: site-specific cleavage of two nucleotides from both 3' ends of viral DNA, and sequence-independent joining of the recessed viral ends to staggered phosphates in a target DNA. Here we investigate human immunodeficiency virus type 1 (HIV-1) IN-DNA interactions using surface plasmon resonance. The results show that IN forms tight complexes both with duplex oligonucleotides that represent the viral DNA ends and with duplex oligonucleotides with an unrelated sequence that represent a target DNA substrate. The IN-DNA complexes are stable in 4.0 M NaCl, or 50% (v/v) methanol, but they are not resistant to low concentrations of SDS, indicating that their stability is highly dependent on structural features of the protein. Divalent metal cofactors exert two distinct effects on the IN-DNA interaction. Mn2+ inhibits IN binding to a model target DNA with the apparent Kd increasing approximately 3-fold in the presence of this cation. On the other hand, Mn2+ (or Mg2+) stimulates the binding of IN to a model viral DNA end, decreasing the apparent Kd of this IN-viral DNA complex approximately 6-fold. Such metal-mediated stimulation of the binding of IN to the viral DNA is totally abolished by substitution of the subterminal conserved CA/GT bp with a GT/CA bp, and is greatly diminished when the viral DNA end is recessed or "pre-processed." IN binds to a viral duplex oligonucleotide whose end was extended with nonviral sequences with kinetics similar to the nonviral model target DNA. This suggests that IN can distinguish the integrated DNA product from the viral donor DNA in the presence of divalent metal ion. Thus, our results show that preferential recognition of viral DNA by HIV-1 IN is achieved only in the presence of metal cofactor, and requires a free, wild-type viral DNA end.     

Minichromosomal DNA replication in the macronucleus of the hypotrichous ciliate Stylonychia lemnae is independent of chromosome-internal sequences

The origins of DNA replication in prokaryotes and eukaryotes are typically defined by cis-acting sequences. However, in ciliates, evidence suggests that the replication of short macronuclear minichromosomes may not require such determinants. In hypotrichous ciliates, macronuclei contain millions of gene-sized minichromosomes, which generally have a single protein-coding region, two short noncoding flanks and, on each end, a short telomere consisting of a double-stranded repeat region and a single-stranded 3' overhang. Electron microscopic studies that showed that replication of minichromosomes initiates at or near telomeres and the discovery of a primase activity synthesizing RNA primers over the whole 3' telomeric overhang in vitro suggested that minichromosome replication starts directly at telomeres. Conversely, many minichromosomes contain an AT-rich, semi-conserved, palindromic sequence motif in their subtelomeric regions and it has been proposed that this motif is involved in regulating minichromosomal replication. To analyze what sequences or structures of the minichromosomes are essential for DNA replication, we stably transfected genetically modified alpha1-tubulin-encoding minichromosomes into the hypotrichous ciliate Stylonychia lemnae. Cotransfection of mutated and control minichromosomes revealed that noncoding regions can be deleted or replaced with unrelated sequences without affecting minichromosome replication efficiency in vegetatively growing cells. Similarly, replacement of the coding region resulted in a minichromosome that was stably maintained in transfected cells at the same high copy number for many months. In contrast, alpha1-tubulin-encoding minichromosomes without telomeres were rapidly lost after transfection. Hence, DNA replication of the alpha1-tubulin-encoding minichromosome does not depend on chromosome-internal sequences but may depend on telomeres.     

Chromatin structure of the potential Z-forming sequence (dT-dG)n X (dC-dA)n. Evidence for an "alternating-B" conformation

The sequence (dT-dG)n X (dC-dA)n is the most abundant purine-pyrimidine dinucleotide repeat in eukaryotic genomes. This sequence and certain others that contain alternating purine-pyrimidine residues have been shown to adopt the left-handed, Z-DNA conformation in vitro when subjected to negative torsional stress or elevated ionic strengths. We have asked whether (dT-dG)n X (dC-dA)n tracts exist in topologically constrained Z-form structures in vivo by examining the chromatin organization of these sequences in cultured mouse cell nuclei. We find that these elements are quantitatively packaged into typical core particles which are embedded in canonical polynucleosomal arrays. In addition, these sequences neither flank nor reside within regions of chromatin that are preferentially sensitive to S1 nuclease. These characteristics suggest that these tracts do not exist predominantly in the Z-form in vivo. Furthermore, employing techniques that permit prominent hybridization to DNA fragments as short as 18 bases, we provide evidence that in vivo, most (dT-dG)n X (dC-dA)n elements instead adopt an "alternating-B" conformation on the nucleosomal surface.