Occurrence of DNA structures which differ from the canonic B-form in sites of highly specific interaction with chromatin proteins

According to the three-dimensional structure of DNase I and the mechanism of its action on linear double-stranded DNA, helix regions in conformations considerably different from the canonical B-form should be resistant to endonucleolysis. A number of DNA sequences specifically bound by nonhistone factors within 5'-flanking regions of the chicken beta A-globin, beta H-globin and c-myc genes are shown to contain short DNase I-resistant DNA domains. Several examples of the occurrence of such DNase I-resistant domains within the sites for high-specific recognition by different proteins are given. The role of the DNA structural polymorphism in site-specific interaction with protein factors is discussed.     

Tetraplex formation of surface-immobilized human telomere sequence probed by surface plasmon resonance using single-stranded DNA binding protein

Many sequences in genomic DNA are able to form unique tetraplex structures. Such structures are involved in a variety of important cellular processes and are emerging as a new class of therapeutic targets for cancers and other diseases. Screening for molecules targeting the tetraplex structure has been explored using such sequences immobilized on solid surfaces. Immobilized nucleic acids, in certain situations, may better resemble the molecules under in vivo conditions. In this report, we studied the formation of tetraplex structure of both the G-rich and C-rich strands of surface-immobilized human telomere sequence by surface plasmon resonance using the single-stranded DNA binding protein from Escherichia coli as probe. We demonstrate how the formation of G-quadruplex and i-motif could be probed under various conditions by this sequence-universal method. Our results also show that immobilization destabilized the tetraplex structure.     

Formation of Single-Stranded DNA during DNA Transformation of Neisseria gonorrhoeae

Neisseria gonorrhoeae is naturally competent for DNA transformation. In contrast to other natural prokaryotic DNA transformation systems, single-stranded donor DNA (ssDNA) has not previously been detected during transformation of N. gonorrhoeae. We have reassessed the physical nature of gonococcal transforming DNA by using a sensitive nondenaturing native blotting technique that detects ssDNA. Consistent with previous analyses, we found that the majority of donor DNA remained in the double-stranded form, and only plasmid DNAs that carried the genus-specific DNA uptake sequence were sequestered in a DNase I-resistant state. However, when the DNA was examined under native conditions, S1 nuclease-sensitive ssDNA was identified in all strains tested except for those bacteria that carried the dud-1 mutation. Surprisingly, ssDNA was also found during transformation of N. gonorrhoeae comA mutants, which suggested that ssDNA was initially formed within the periplasm.     

DNA tetraplex formation studied with fluorescence resonance energy transfer.

It is emerging that DNA tetraplexes are pivotal for many major cellular processes, and techniques that assess their structure and nature to the point are under development. Here we show how the structural conversion of largely unstructured single-stranded DNA molecules into compact intrastrand DNA tetraplexes can be monitored by fluorescence resonance energy transfer. We recently reported that intrastrand tetraplex formation takes place in a nuclease hypersensitive element upstream of the human c-myc proto-oncogene. Despite the highly repetitive guanine-rich sequence of the hypersensitive element, fluorescence resonance energy transfer measurements indicate that only one well defined tetraplex structure forms therein. The proposed structure, which is specifically stabilized by potassium ions in vitro, has a core of three stacked guanine tetrads that is capped by two intrastrand A-T base pairs.     

Circular dichroism spectroscopy reveals invariant conformation of guanine runs in DNA

We demonstrate that the characteristic circular dichroism (CD) features of the parallel-stranded DNA tetraplex of d(G4), especially the strong band at 260 nm, are characteristic for the B and A forms of the antiparallel duplex of d(C4G4). Hence, this band evidently originates from intrastrand guanine-guanine stacking, which is therefore very similar in the duplex and tetraplex DNA. In addition, the same type of the CD spectrum is provided by the ordered single strand of d(GA)10. This observation suggests that the ordered single strand of d(GA)10 is stabilized by a core of guanines stacked like in the parallel tetraplex. This view is used to start the modeling of the molecular structure of the ordered d(GA)10 single strand. Our studies suggest that guanine itself is strong enough to stabilize various secondary structures of DNA, which is a property relevant to thinking about the origin and evolution of molecular replicators.     

Mobile Element Insertions Causing Mutations in the Drosophila Suppressor of Sable Locus Occur in Dnase I Hypersensitive Subregions of 5''-Transcribed Nontranslated Sequences

The locations of 16 mobile element insertions causing mutations at the Drosophila suppressor of sable [su(s)] locus were determined by restriction mapping and DNA sequencing of the junction sites. The transposons causing the mutations are: P element (5 alleles), gypsy (3 alleles), 17.6, HMS Beagle, springer, Delta 88, prygun, Stalker, and a new mobile element which was named roamer (2 alleles). Four P element insertions occur in 5' nontranslated leader sequences, while the fifth P element and all 11 non-P elements inserted into the 2053 nucleotide, 5'-most intron that is spliced from the 5' nontranslated leader approximately 100 nucleotides upstream of the translation start. Fifteen of the 16 mobile elements inserted within a approximately 1900 nucleotide region that contains seven 100-200-nucleotide long DNase I-hypersensitive subregions that alternate with DNase I-resistant intervals of similar lengths. The locations of these 15 insertion sites correlate well with the roughly estimated locations of five of the DNase I-hypersensitive subregions. These findings suggest that the features of chromatin structure that accompany gene activation may also make the DNA susceptible to insertion of mobile elements.     

DNase Specific for Uracil-Containing Bacteriophage DNA

A DNase from Bacillus subtilis which specifically hydrolyzes native DNA of phage PBS 1 has been purified and characterized. The mode of action of the enzyme is endonucleolytic, yielding deoxyuridine and oliogonucleotides of various sizes. The primary site of enzymatic attack is deoxyuridylic acid in the DNA. A mild nitrous acid treatment of thymine-containing thymus DNA, which deaminates 30% of the cytosine residues, renders the DNA susceptible to the DNase. Nicked DNA from coli phage T5 and hydroxymethyluracil-containing DNA from phage PBS 15 are not sensitive to this DNase.     

Tetraplex structure formation in the thrombin-binding DNA aptamer by metal cations measured by vibrational spectroscopy

Formation of intramolecular tetraplex structures by the thrombin-binding DNA aptamer (TBA) in the presence of K(+), Pb(2+), Ba(2+), Sr(2+) and Mn(2+) has been studied by vibrational spectroscopy. All tetraplex structures contain G-G Hoogsteen type base pairing, both C2'endo/anti and C2'endo/syn deoxyguanosine glycosidic conformations and local B like form DNA phosphate geometries. Addition of Pb(2+) ions modifies the structure by interacting at the level of the guanine carbonyl groups. The very important downshift of the guanine C6=O6 carbonyl vibration mode in the TBA spectrum induced by the addition of one Pb(2+) ion per TBA molecule is in agreement with a localization of the metal ion between both guanine quartets. FTIR melting experiments show an important stabilization of the tetraplex structure upon addition of Pb(2+) ions (DeltaT = 15 degrees C). This strong interaction of lead cations may be correlated with a change in the geometry of the cage formed by the two guanine quartets. A similar but weaker effect is observed for barium and strontium cations.     

Rec A-independent homologous recombination induced by a putative fold-back tetraplex DNA

We have recently reported that a GC-rich palindromic repeat sequence presumably adopts a stable fold-back tetraplex DNA structure under supercoiling. To establish the biological significance of this structure, we inserted this sequence between two direct repeat sequences, separated by 200 bp, in a plasmid. We then investigated the effect of this sequence on homologous recombination events. Here we report that the putative fold-back DNA tetraplex structure induces homologous recombination between direct repeat sequences. Interestingly, this recombination event is independent of recA, a major driving force for homologous recombination. We think that the fold-back structure forces the repeat sequences to come into close proximity and therefore leads to strand exchange. Although triplex-induced recombination has been well documented, our results for the first time directly establish the potential of a tetraplex structure to induce recA-independent homologous recombination in vivo. This finding might have a significant implication for site-directed gene deletion in the context of the correction of genetic defects.     

Sedimentation velocity properties of catenated DNA molecules from HeLa cell mitochondria

Catenated molecules of closed circular DNA have been isolated from the mitochondrial DNA of HeLs cells. The sedimentation coefficients of several purified species have been investigated. The catenated dimer, made up of two interlocked duplex circles, sediments at 51 S in its superhelical (closed) form. Treatment with pancreatic DNase to relax the duplex circles converts the 51 S doubly closed dimer to a 42 S singly open species, then to a 36 S doubly open catenated dimer. The triply closed trimer sediments at 63 S and is converted to a 45 S triply open form by DNase. Electron microscopy of the DNA samples before and after DNase treatment shows that under the conditions used DNase does not change the catenated nature of the DNA. The measured sedimentation coefficients, have been compared with those estimated from previously proposed correlations of sedimentation coefficient and molecular weight, and with the sedimentation coefficients for catenated DNA presented by Wang. When all the interlocked circles in a catenane are relaxed, the DNA sediments about 5–10% faster than a relaxed multiple-length circular molecule of the same molecular weight. The sedimentation coefficient, 36 S, of the fully relaxed catenated dimer is 1.4 times that of the relaxed monomer.     

The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites.

The structure of the major human apurinic/ apyrimidinic endonuclease (HAP1) has been solved at 2.2 A resolution. The enzyme consists of two symmetrically related domains of similar topology and has significant structural similarity to both bovine DNase I and its Escherichia coli homologue exonuclease III (EXOIII). A structural comparison of these enzymes reveals three loop regions specific to HAP1 and EXOIII. These loop regions apparently act in DNA abasic site (AP) recognition and cleavage since DNase I, which lacks these loops, correspondingly lacks AP site specificity. The HAP1 structure furthermore suggests a mechanism for AP site binding which involves the recognition of the deoxyribose moiety in an extrahelical conformation, rather than a 'flipped-out' base opposite the AP site.     

Thermolabile in vivo DNA-binding activity associated with a protein encoded by mutants of herpes simplex virus type 1.

The major DNA-binding protein encoded by several temperature-sensitive mutants of herpes simplex virus type 1 was thermolabile for binding to intracellular viral DNA. The ability of DNase I to release this protein from isolated nuclei was used as a measure of the amount of protein bound to viral DNA. This assay was based upon our previous observation that the fraction of herpesviral DNA-binding protein which can be eluted from nuclei with DNase I represents proteins associated with progeny viral DNA (D. M. Knipe and A. E. Spang, J. Virol. 43:314-324, 1982). In this study, we found that several temperature-sensitive mutants encoded proteins which rapidly chased from a DNase I-sensitive to a DNase I-resistant nuclear form upon shift to the nonpermissive temperature. We interpret this change in DNase I sensitivity to represent the denaturation of the DNA-binding site at the nonpermissive temperature and the association with the nuclear framework via a second site on the protein. The DNA-binding activity measured by the DNase I sensitivity assay represents an important function of the protein in viral replication because three of five mutants tested were thermolabile for this activity. A fourth mutant encoded a protein which did not associate with the nucleus at the nonpermissive temperature and therefore would not be available for DNA binding in the nucleus. We also present supportive evidence for the binding of the wild-type protein to intracellular viral DNA by showing that a monoclonal antibody coprecipitated virus-specific DNA sequences with the major DNA-binding protein.     

Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1).

Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long-patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap-filling. XPG, a related nuclease, could not substitute for DNase IV. The long-patch BER pathway was largely dependent on DNA polymerase beta in cell extracts, but the reaction could be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV.     

Tetraplex structure of fission yeast telomeric DNA and unfolding of the tetraplex on the interaction with telomeric DNA binding protein Pot1

To understand the regulation mechanism of fission yeast telomeric DNA, we analysed the structural properties of Gn: d(GnTTAC) (n=2-6) and 4Gn: d(GnTTAC)4 (n=3 and 4), and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). G4, G5 and G6 formed a parallel tetraplex in contrast with no tetraplex formation by G2 and G3. Also, 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The variety of tetraplex structures was governed by the number of consecutive guanines in a single copy and the number of repeats. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. The interaction with mutant Pot1DBD proteins revealed that the ability to unfold the antiparallel tetraplex was strongly correlated with the specific binding affinity for the single-stranded telomeric DNA. The result suggests that the decrease in the free single strand upon the complex formation with Pot1DBD may shift the equilibrium from the tetraplex to the single strand, which may cause the tetraplex unfolding. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.     

Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition,catalysis, and metal ion dependence.

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.