The Z-DNA motif d(TG)30 promotes reception of information during gene conversion events while stimulating homologous recombination in human cells in culture.

Tracts of the alternating dinucleotide polydeoxythymidylic-guanylic [d(TG)].polydeoxyadenylic-cytidylic acid [d(AC)], present throughout the human genome, are capable of readily forming left-handed Z-DNA in vitro. We have analyzed the effects of the Z-DNA motif d(TG)30 upon homologous recombination between two nonreplicating plasmid substrates cotransfected into human cells in culture. In this study, the sequence d(TG)30 is shown to stimulate homologous recombination up to 20-fold. Enhancement is specific to the Z-DNA motif; a control DNA fragment of similar size does not alter the recombination frequency. The stimulation of recombination is observed at a distance (237 to 1,269 base pairs away from the Z-DNA motif) and involves both gene conversion and reciprocal exchange events. Maximum stimulation is observed when the sequence is present in both substrates, but it is capable of stimulating when present in only one substrate. Analysis of recombination products indicates that the Z-DNA motif increases the frequency and alters the distribution of multiple, unselected recombination events. Specifically designed crosses indicate that the substrate containing the Z-DNA motif preferentially acts as the recipient of genetic information during gene conversion events. Models describing how left-handed Z-DNA sequences might promote the initiation of homologous recombination are presented.     

Sequence-specific interactions between a cellular DNA-binding protein and the simian virus 40 origin of DNA replication.

The core origin of simian virus 40 (SV40) DNA replication is composed of a 64-base-pair sequence encompassing T-antigen-binding site II and adjacent sequences on either side. A 7-base-pair sequence to the early side of T-antigen-binding site II which is conserved among the papovavirus genomes SV40, BK, JC, and SA12 was recently shown to be part of a 10-base-pair sequence required for origin activity (S. Deb, A.L. DeLucia, C.-P. Baur, A. Koff, and P. Tegtmeyer, Mol. Cell. Biol. 6:1663-1670, 1986), but its functional role was not defined. In the present report, we have used gel retention assays to identify a monkey cell factor that interacts specifically with double-stranded DNA carrying this sequence and also binds to single-stranded DNA. DNA-protein complexes formed with extracts from primate cells are more abundant and display electrophoretic mobilities distinct from those formed with rodent cell extracts. The binding activity of the factor on mutant templates is correlated with the replication activity of the origin. The results suggest that the monkey cell factor may be involved in SV40 DNA replication.     

Left-handed DNA and the synaptic pairing reaction promoted by Ustilago rec1 protein

Left-handed Z-DNA binds tightly to Ustilago rec1 protein. The binding reaction is strongly dependent on ATP, but complexes formed are rapidly dissociated by ADP. The parallel between the kinetics of Z-DNA binding and the synaptic pairing reaction leading to paranemic joint molecules suggests that formation of nascent heteroduplex structures in recombination is coupled with formation of left-handed Z-like DNA on the protein. Equilibrium and kinetic studies show that rec1 protein appears to have a strong Z-DNA binding site that binds Z-DNA 75 times tighter than the B form of the DNA. We propose that DNA with a structure approximated best by a left-handed Z-DNA conformation is a key intermediate in homologous pairing promoted by rec1 protein.     

Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro.

We have already reported that the cloned mouse DNA fragment (pMU65) could replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro (H. Ariga, Z. Tsuchihashi, M. Naruto, and M. Yamada, Mol. Cell. Biol. 5:563-568, 1985). The plasmid p65-tk, containing the thymidine kinase (tk) gene of herpes simplex virus and the BglII-EcoRI region of pMU65 homologous to the simian virus 40 origin of DNA replication, was constructed. The p65-tk persisted episomally in tk+ transformants after the transfection of p65-tk into mouse FM3Atk- cells. The copy numbers of p65-tk in FM3Atk+ cells were 100 to 200 copies per cell. Furthermore, the p65-tk replicated semiconservatively, and the initiation of DNA replication started from the mouse DNA sequences when the replicating activity of p65-tk was tested in the in vitro DNA replication system developed from the FM3A cells. These results show that a 2.5-kilobase fragment of mouse DNA contains the autonomously replicating sequences.     

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.     

Simian virus 40 large tumor antigen on replicating viral chromatin: tight binding and localization on the viral genome

Pulse-labeled simian virus 40 (SV40) chromatin as well as uniformly labeled viral chromatin are immunoprecipitable by an SV40-specific tumor antiserum and therefore contain bound tumor antigen (T antigen). Single-stranded calf thymus DNA, immobilized on cellulose, competes effectively for T antigen binding with uniformly labeled nonreplicating, but not with pulse-labeled replicating, chromatin. Furthermore, T antigen dissociates in 0.5 M NaCl from nonreplicating chromatin and from purified SV40 DNA, whereas most T antigen remains associated with replicating chromatin even in the presence of 1.2 to 1.5 M NaCl. We used filtration through DNA-cellulose columns and treatment with high salt to prepare pulse-labeled immunoreactive viral chromatin. The viral DNA was digested before, and in other experiments after, immunoprecipitation with the restriction endonuclease HindIII. We found that SV40 DNA sequences, most probably representing the entire genome, remain in the immunoprecipitate after HindIII digestion, indicating an association of T antigen with origin-distal sections of replicating viral DNA. The results suggest that T antigen in replicating chromatin may be bound to regions close to replicating points. We performed control experiments with in vitro-formed complexes of T antigen and SV40 DNA. When these complexes were immunoprecipitated and HindIII digested we found, in agreement with previous studies, that only the origin containing the HindIII C fragment carried bound T antigen.     

Induction of Duplication Reversion in Human Fibroblasts, by Wild-Type and Mutated Sv40 T Antigen, Covaries with the Ability to Induce Host DNA Synthesis

Intrachromosomal homologous recombination, manifest as reversion of a 14-kbp duplication in the hypoxanthine phosphoribosyl transferase (HPRT) gene, is elevated in human cells either stably transformed or transiently transfected by the SV40 (simian virus 40) large T antigen gene. Following introduction of wild-type SV40, or any of several T-antigen point mutations in a constant SV40 background, we observed a strong correlation between the stimulation of chromosomal recombination and induction of host-cell DNA synthesis. Moreover, inhibitors of DNA replication (aphidicolin and hydroxyurea) suppress SV40-induced homologous recombination to the extent that they suppress DNA synthesis. Stable integration of plasmids encoding T antigen also augments homologous recombination, which is suppressed by aphidicolin. We infer that the mechanism by which T antigen stimulates homologous recombination in human fibroblasts involves DNA replicative synthesis.     

Left-handed Z-DNA binding by the recA protein of Escherichia coli

recA binding to left-handed Z-DNA was measured using nitrocellulose filter binding assays with four DNA polymers with defined nucleotide sequences and four recombinant plasmids. Two to 7-fold preferential binding of recA to Z-DNA polymers was observed. Left-handed Z-DNA polymer binding by recA required ATP or its nonhydrolyzable analog, ATP(gamma S), while ADP inhibited binding. Complex formation with both B- and Z-forms was influenced by polymer length; recA bound longer DNAs better. recA binding to recombinant plasmids containing supercoil-stabilized Z-DNA was essentially similar to that found for the control vector; thus, no preferential binding of recA to the Z-form was observed. Comparative experiments with the rec1 protein of Ustilago maydis and the Escherichia coli recA protein were performed. In our hands, recA and rec1 have a similar capacity for binding left-handed Z-DNA polymers and for binding recombinant plasmids containing B- and/or Z-regions. recA contains a left-handed Z-DNA-stimulated ATPase activity. This activity differs from the right-handed B-DNA-stimulated activity since it is less sensitive to increasing pH. The kinetics of ATP hydrolysis in B-DNA/Z-DNA mixing experiments showed that the turnover of the Z-DNA recA complex was slower than for B-DNA suggesting that left-handed Z-DNA is more stably bound by recA. Our results are consistent with the postulate that left-handed Z-DNA is involved in genetic recombination.     

Location of B- and Z-DNA in the chromosomes of a primitive eukaryote dinoflagellate

The usual conformation of DNA is a right-handed double helix (B-DNA). DNA with stretches of alternating purine-pyrimidine (G-C or A-T) can form a left-handed helix (Z-DNA). The transition B----Z, facilitated by the presence of divalent cations, cytosine methylation, or constraints on DNA such as superhelicity may play a role in the regulation of gene expression and/or in DNA compaction (Zarling, D. A., D. J. Arndt-Jovin, M. Robert-Nicoud, L. P. McIntosh, R. Tomae, and T. M. Jovin. 1984. J. Mol. Biol. 176:369-415). Divalent cations are also important in the structure of the quasi-permanently condensed chromosomes of dinoflagellate protists (Herzog, M., and M.-O. Soyer. 1983. Eur. J. Cell Biol. 30:33-41) which also have superhelicity in their DNA. The absence of histones in dinoflagellate chromosomes suggest that the search for Z-DNA sequences might be fruitful and could provide one indication of the physiological role of this particular DNA conformation. We report a complete immunofluorescent and immunogold analysis of the nuclei of the dinoflagellate Prorocentrum micans E. using monoclonal and polyclonal anti-B and anti-Z-DNA antibodies. Positive labeling was obtained with immunofluorescence using squash preparations and cryosections, both of which showed the intranuclear presence of the two DNA conformations. In ultrathin sections of aldehyde-prefixed, osmium-fixed, and epoxy-embedded cells, we have localized B-DNA and Z-DNA either with single or double immunolabeling using IgG labeled with 5- and 7-nm gold particles, respectively. Chromosomal nucleofilaments of dividing or nondividing chromosomes, as seen in ultrathin sections in their arch-shaped configuration, are abundantly labeled with anti-B-DNA antibody. Extrachromosomal anti-B-DNA labeling is also detected on the nucleoplasm that corresponds to DNA loops; we confirm the presence of these loops previously described external to the chromosomes (Soyer, M.-O., and O. K. Haapala. 1974. Chromosoma (Berl.). 47:179-192). B labeling is also visible in the nucleolus organizer region (NOR) and in the fibrillo-granular area (containing transcribing rDNA) of the nucleolus. Z-DNA was localized in limited areas inside the chromosomes, often at the periphery and near the segregation fork of dividing chromosomes. In the nucleolus, Z-DNA is observed only in the NOR area and never in the fibrillo-granular area. For both types of antibody experiments, controls using gold-labeled IgG without primary antibody were negative. A quantitative evaluation of the distribution of the gold-labeled IgG and a parametric test support the validity of these experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Repair of double-strand breaks by homologous recombination in mismatch repair-defective mammalian cells

Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93-101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2(-/-) cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.     

Mapping of replication initiation sites in human ribosomal DNA by nascent-strand abundance analysis.

New techniques for mapping mammalian DNA replication origins are needed. We have modified the existing nascent-strand size analysis technique (L. Vassilev and E.M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989) to provide an independent means of studying replication initiation sites. We call the new method nascent-strand abundance analysis. We confirmed the validity of this method with replicating simian virus 40 DNA as a model. We then applied nascent-strand abundance and nascent-strand size analyses to mapping of initiation sites in human (HeLa) ribosomal DNA (rDNA), a region previously examined exclusively by two-dimensional gel electrophoresis methods (R.D. Little, T.H.K. Platt, and C.L. Schildkraut, Mol. Cell. Biol. 13:6600-6613, 1993). Our results partly confirm those obtained by two-dimensional gel electrophoresis techniques. Both studies suggest that replication initiates at relatively high frequency a few kilobase pairs upstream of the transcribed region and that many additional low-frequency initiation sites are distributed through most of the remainder of the ribosomal DNA repeat unit.     

The distribution of large T antigen in simian virus 40 nucleoprotein complexes

Simian virus 40 large T antigen binds to two types of nucleoprotein complexes from lytically infected cells: those containing replicating virus DNA (100S complexes) and those containing nonreplicating virus DNA (70S complexes). Analysis by agarose gel electrophoresis showed that replicating DNA was found exclusively in 100S complexes, although these complexes also contained large amounts of form I and form II DNA. In contrast, no replicating DNA was found in 70S complexes, and pulse-labeled DNA in these complexes migrated as form I and form II DNA that presumably had recently completed replication. Immunoprecipitation and gel electrophoresis showed that large T antigen was associated with both types of complexes. From 21 to 62% of replicating DNA in 100S complexes was bound to T antigen. Our estimates indicated, however, that more than three-fourths of the DNA molecules in 100S complexes were nonreplicating and unassociated with T antigen. In 70S complexes, 12 to 31% of pulse-labeled DNA was bound to T antigen, but because there were more DNA molecules in the 70S complexes, they contained a greater absolute amount of T antigen.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

Bromination stabilizes poly(dG-dC) in the Z-DNA form under low-salt conditions

Using circular dichroism studies, Pohl & Jovin (1972) [Pohl, F.M., & Jovin, T.M. (1972) J. Mol. Biol. 67, 375-396] demonstrated that poly(dG-dC) undergoes a salt-dependent conformational change characterized by a spectral inversion. The low-salt form corresponds to the right-handed B form of DNA and the high-salt form to the left-handed Z-DNA helix. Modification of poly(dG-dC) by adding bromine atoms to the C8 position of guanine and the C5 position of cytosine residues stabilized this polymer in the Z-DNA form under low-salt conditions. The guanine residues were found to be twice as reactive as the cytosine residues. With a modification of 38% Br8G and 18% Br5C, the polymers formed a stable Z-DNA helix under physiological conditions. The bromination produced spectroscopic features very similar to poly(dG-dC) in 4 M NaCl. However, bromination did not freeze the Z structure as was shown by ethidium bromide intercalation studies. Addition of the dye favored an intercalated B-DNA form. The conversion of B- to Z-DNA leads to profound conformational changes which were also seen by a reduced insensitivity to various exo- and endonucleases. Comparative studies showed that the brominated polymers have a high affinity to nitrocellulose filters. In 1 M NaCl, there was virtually no binding of B-DNA, but a substantial binding of Z-DNA was found even at rather low levels of bromination.(ABSTRACT TRUNCATED AT 250 WORDS)