DNA length tunes the fluidity of DNA-based condensates

Living organisms typically store their genomic DNA in a condensed form. Mechanistically, DNA condensation can be driven by macromolecular crowding, multivalent cations, or positively charged proteins. At low DNA concentration, condensation triggers the conformational change of individual DNA molecules into a compacted state, with distinct morphologies. Above a critical DNA concentration, condensation goes along with phase separation into a DNA-dilute and a DNA-dense phase. The latter DNA-dense phase can have different material properties and has been reported to be rather liquid-like or solid-like depending on the characteristics of the DNA and the solvent composition. Here, we systematically assess the influence of DNA length on the properties of the resulting condensates. We show that short DNA molecules with sizes below 1 kb can form dynamic liquid-like assemblies when condensation is triggered by polyethylene glycol and magnesium ions, binding of linker histone H1, or nucleosome reconstitution in combination with linker histone H1. With increasing DNA length, molecules preferentially condense into less dynamic more solid-like assemblies, with phage λ-DNA with 48.5 kb forming mostly solid-like assemblies under the conditions assessed here. The transition from liquid-like to solid-like condensates appears to be gradual, with DNA molecules of roughly 1–10 kb forming condensates with intermediate properties. Titration experiments with linker histone H1 suggest that the fluidity of condensates depends on the net number of attractive interactions established by each DNA molecule. We conclude that DNA molecules that are much shorter than a typical human gene are able to undergo liquid-liquid phase separation, whereas longer DNA molecules phase separate by default into rather solid-like condensates. We speculate that the local distribution of condensing factors can modulate the effective length of chromosomal domains in the cell. We anticipate that the link between DNA length and fluidity established here will improve our understanding of biomolecular condensates involving DNA.     

Coupling into the base pair stack is necessary for DNA-mediated electrochemistry

The electrochemistry of DNA films modified with different redox probes linked to DNA through saturated and conjugated tethers was investigated. Experiments feature two redox probes bound to DNA on two surfaces: anthraquinone (AQ)-modified uridines incorporated into thiolated DNA on gold (Au) and 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)-modified uridines in pyrene-labeled DNA on highly oriented pyrolytic graphite (HOPG). The electrochemistry of these labels when incorporated into DNA has been examined in DNA films containing both well matched and mismatched DNA. DNA-mediated electrochemistry is found to be effective for the TEMPO probe linked with an acetylene linker but not for a saturated TEMPO connected through an ethylenediamine linker. For the AQ probe, DNA-mediated electrochemistry is found with an acetylene linker to uridine but not with an alkyl chain to the 5' terminus of the oligonucleotide. Large electrochemical signals and effective discrimination of intervening base mismatches are achieved for the probes connected through the acetylene linkages, while probes connected through saturated linkages exhibit small electrochemical signals associated only with direct surface to probe charge transfer and poor mismatch discrimination. Thus DNA electrochemistry with these probes is dramatically influenced by the chemical nature of their linkage to DNA. These results highlight the importance of effective coupling into the pi-stack for long-range DNA-mediated electrochemistry.     

Gene delivery into hepatocytes with the preS/liposome/DNA system

Gene delivery into human hepatocytes remains a critical issue for the development of liver-directed gene therapy. Gene delivery based on non-viral vectors is an attractive approach relative to viral vectors. In this report, novel delivery system of preS/liposome/DNA virus-like particle (VLP) was developed for gene transfection into hepatocytes in vivo and in vitro. Plasmid pCMVbeta, expressing beta-galactosidase, was encapsulated with cationic liposome, and then the histidine-tagged preS domain of hepatitis B virus was coated on the surface of liposome/DNA to form preS/liposome/ DNA VLP. Transfection efficiencies of preS/liposome/DNA, liposome/DNA, naked DNA and preS were analyzed using several different human cell lines. The highest transfection efficiency was found using preS/liposome/DNA VLP as the transfection reagent in human hepatocyte (HH) cell line. Results show that preS domain of hepatitis B virus coated on liposome/DNA can be used for highly efficient gene transfection into human hepatocytes. Moreover, the target characteristic of preS/liposome/DNA was analyzed in vivo. After preS/liposome/DNA VLP was injected into immunocompromised (Nude) mice via the tail vein, most of beta-galactosidase was expressed in the liver; however, no significant target expression was found with the injection of liposome/ DNA or naked DNA. Our results show that preS/liposome/DNA VLP can be used as a novel liver-specific gene delivery system.     

FITC示踪在优化小麦花粉管通道转化方法中的应用

利用FITC(fluorescein is othiocyanate,异硫氰酸荧光素)标记的外源DNA对小麦进行了花粉管通道法转化,在荧光显微镜下观察了外源DNA进入小麦胚囊的情况,并初步研究了转化时间及转化溶液组成对DNA进入胚囊效率的影响。结果表明,外源DNA沿着花粉管生长过程中形成的花粉管通道进入胚囊;授粉45 min和60min进行转化外源DNA进入胚囊的几率较大,因此在此段时间开始转化较为合适;相对于TE缓冲液,将0.05%Silwet L-77和5%蔗糖作为转化溶液可以缩短DNA进入胚囊的时间,但不能提高外源DNA进入胚囊的几率。研究表明,使用FITC标记观察外源DNA进入胚囊效率的方法,可简便有效地应用于花粉管通道法转化条件的初步优化。     

DNA synthesis in the elongating nondividing cells of the lentil epicotyl and its promotion by gibberellin

Two-day-old lentil seedlings, (Lens culinaris Med.) were incubated for a 48-hour period with and without gibberellin (GA) in the presence and absence of 5-fluorodeoxyuridine (FUDR). The number of cells per epicotyl did not increase during this period. Growth of the epicotyl was thus due to cell elongation alone.

The elongating cells of this tissue synthesized DNA. GA promoted and FUDR inhibited cell elongation, DNA synthesis, and RNA synthesis in the tissue.

FUDR promoted uptake of thymidine and thymidine incorporation into cellular DNA, presumably by inhibiting synthesis of endogenous thymidine. Presence of GA promoted thymidine incorporation into cellular DNA and uridine incorporation into cellular RNA. In either case, there was no effect on the uptake of the precursor into the tissue.

Fractionation of thymidine-labeled nucleic acids on a MAK column showed that thymidine was exclusively incorporated into the DNA fraction. Presence of GA promoted thymidine incorporation into this fraction and also increased the amount of ribosomal RNA.

The data provide direct evidence for the conclusion that DNA synthesis is necessary for elongation of certain plant cells.

     

Accelerated microsomal DNA synthesis under the influence of xenobiotics and chemical carcinogens]

Injection of 3,4-benz(a)pyrene, methyl nitrosourea and phenobarbital into healthy mice of the C3HA line results in a rapid, sharp increase of [14C]-thymidine incorporated into liver microsomal DNA, accompanied by a suppression of nuclear DNA synthesis. In the liver of neoplastic mice and in the ascite cells of hepatoma 22A the system of microsomal DNA synthesis was insensitive to the injection of methyl nitrosourea. Cycloheximide and puromycin, which strongly inhibited nuclear DNA synthesis, had no effect on the synthesis of microsomal DNA. Stimulation of [14C]-thymidine incorporation into microsomal DNA after injection of methyl nitrosourea and 3,4-benz(a)pyrene may be accounted for not only by an increase of the DNA reparation processes, since caffeine, the inhibitor of post-replicatory reparation of DNA, did not eliminate the induction of microsomal DNA synthesis in the liver. Hydroxyurea in combination with methyl nitrosourea and phenobarbital significantly suppressed the synthesis of nuclear DNA in the liver and did not affect the synthesis of mtDNA; the stimulating effects of these inducers on the synthesis of microsomal DNA was thereby removed. This is indicative of independence of synthesis of microsomal DNA on that of nuclear DNA and mtDNA. Different specific radioactivities of microsomal, nuclear and mtDNAs in the regenerating mouse liver on the 5th, 10th and 15th post-hepatectomy days may be due to different metabolic stability of these DNAs. A possible role of microsomal DNA as a xenobiotic system component is discussed.     

Visualizing the assembly and disassembly mechanisms of the MuB transposition targeting complex

MuB, a protein essential for replicative DNA transposition by the bacteriophage Mu, is an ATPase that assembles into a polymeric complex on DNA. We used total internal reflection fluorescence microscopy to observe the behavior of MuB polymers on single molecules of DNA. We demonstrate that polymer assembly is initiated by a stochastic nucleation event. After nucleation, polymer assembly occurs by a mechanism involving the sequential binding of small units of MuB. MuB that bound to A/T-rich regions of the DNA assembled into large polymeric complexes. In contrast, MuB that bound outside of the A/T-rich regions failed to assemble into large oligomeric complexes. Our data also show that MuB does not catalyze multiple rounds of ATP hydrolysis while remaining bound to DNA. Rather, a single ATP is hydrolyzed, then MuB dissociates from the DNA. Finally, we show that "capping" of the enhanced green fluorescent protein-MuB polymer ends with unlabeled MuB dramatically slows, but does not halt, dissociation. This suggests that MuB dissociation occurs through both an end-dependent mechanism and a slower mechanism wherein subunits dissociate from the polymer interior.     

A defined structure of the 30 nm chromatin fibre which accommodates different nucleosomal repeat lengths

Earlier work on the condensation of chromatins of different repeat lengths into the 30 nm fibre has been surveyed and it is shown that the external geometry of the fibre must be the same for all the chromatins. This can only be fitted by a helical coiling of nucleosomes into a solenoid with the linker DNA disposed internally. On this basis, various models were calculated and compared with published electric dichroism data. The only good fit is found with a 'reverse-loop' model, where the linker DNA forms a complete turn into the hole of the solenoid, of opposite hand to the nucleosomal DNA superhelix. This gives a topological linking number of one per nucleosome and would resolve the 'linking number paradox' if the DNA screw is the same in chromatin as in solution. The feasibility of a reverse-loop for short linkers (down to 15 base pairs) was investigated by model building and kinks of approximately 120 degrees into both DNA grooves are described, which will allow such packing. There will, however, be a 'forbidden' range for the linker DNA length, between approximately 1 and 14 bp, corresponding to nucleosomal repeats of 163 and 176 bp.     

In vitro HIV-1 selective integration into the target sequence and decoy-effect of the modified sequence

Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5'-(GTCCCTTCCCAGT)(n)(ACTGGGAAGGGAC)(n)-3', and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA.     

Structural insights into the retroviral DNA integration apparatus

Retroviral replication depends on successful integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 3' viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes.     

Modulation of the viral ATPase activity by the portal protein correlates with DNA packaging efficiency

DNA packaging in tailed bacteriophages and herpesviruses requires assembly of a complex molecular machine at a specific vertex of a preformed procapsid. As in all these viruses, the DNA translocation motor of bacteriophage SPP1 is composed of the portal protein (gp6) that provides a tunnel for DNA entry into the procapsid and of the viral ATPase (gp1-gp2 complex) that fuels DNA translocation. Here we studied the cross-talk between the components of the motor to control its ATP consumption and DNA encapsidation. We showed that gp6 embedded in the procapsid structure stimulated more than 10-fold the gp2 ATPase activity. This stimulation, which was significantly higher than the one conferred by isolated gp6, depended on the presence of gp1. Mutations in different regions of gp6 abolished or decreased the gp6-induced stimulation of the ATPase. This effect on gp2 activity was observed both in the presence and in the absence of DNA and showed a strict correlation with the efficiency of DNA packaging into procapsids containing the mutant portals. Our results demonstrated that the portal protein has an active control over the viral ATPase activity that correlates with the performance of the DNA packaging motor.     

Characterization of the DNA binding activity of stable RecA-DNA complexes. Interaction between the two DNA binding sites within RecA helical filaments

The DNA-binding, annealing and recombinational activities of purified RecA-DNA complexes stabilized by ATP gamma S (a slowly hydrolysable analog of ATP) are described. Electrophoretic analysis, DNase protection experiments and observations by electron microscopy suggest that saturated RecA complexes formed with single- or double-stranded DNA are able to accommodate an additional single strand of DNA with a stoichiometry of about one nucleotide of added single-stranded DNA per nucleotide or base-pair, respectively, of DNA resident in the complex. This strand uptake is independent of complementarity or homology between the added and resident DNA molecules. In the complex, the incoming and resident single-stranded DNA molecules are in close proximity as the two strands can anneal in case of their complementarity. Stable RecA complexes formed with single-stranded DNA bind double-stranded DNA efficiently when the added DNA is homologous to the complexed strand and then initiate a strand exchange reaction between the partner DNA molecules. Electron microscopy of the RecA-single-stranded DNA complexes associated with homologous double-stranded DNA suggests that a portion of duplex DNA is taken into the complex and placed in register with the resident single strand. Our experiments indicate that both DNA binding sites within RecA helical filaments can be occupied by either single- or double-stranded DNA. Presumably, the same first DNA binding site is used by RecA during its polymerization on single- or double-stranded DNA and the second DNA binding site becomes available for subsequent interaction of the protein-saturated complexes with naked DNA. The way by which additional DNA is taken into RecA-DNA complexes shows co-operative character and this helps to explain how topological problems are avoided during RecA-mediated homologous recombination.     

Probing the effects of DNA-protein interactions on DNA hole transport: the N-terminal histone tails modulate the distribution of oxidative damage and chemical lesions in the nucleosome core particle

The ability of DNA to transport positive charges, or holes, over long distances is well-established, but the mechanistic details of how this process is influenced by packaging into DNA-protein complexes have not been fully delineated. In eukaryotes, genomic DNA is packaged into chromatin through its association with the core histone octamer to form the nucleosome core particle (NCP), a complex whose structure can be modulated through changes in the local environment and the histone proteins. Because (i) varying the salt concentration and removing the histone tails influence the structure of the NCP in known ways and (ii) previous studies have shown that DNA hole transport (HT) occurs in the nucleosome, we have used our previously described 601 sequence NCPs to test the hypothesis that DNA HT dynamics can be modulated by structural changes in a DNA-protein complex. We show that at low salt concentrations there is a sharp increase in long-range DNA HT efficiency in the NCP as compared to naked DNA. This enhancement of HT can be negated by either removal of the histone tails at low salt concentrations or disruption of the interaction of the packaged DNA and the histone tails by increasing the buffer's ionic strength. Association of the histone tails with 601 DNA at low salt concentrations shifts the guanine damage spectrum to favor lesions like 8-oxoguanine in the NCP, most likely through modulation of the rate of the reaction of the guanine radical cation with oxygen. These experimental results indicate that for most genomic DNA, the influence of DNA-protein interactions on DNA HT will depend strongly on the level of protection of the DNA nucleobases from oxygen. Further, these results suggest that the oxidative damage arising from DNA HT may vary in different genomic regions depending on the presence of either euchromatin or heterochromatin.     

Effect of damage to early, middle, and late-replicating DNA on progress through the S period in Chinese hamster ovary cells

In order to explain sequential replication of DNA in eukaryotic cells, the duplication of a given bank of replicons is proposed to be initiated by specific events coupled to synthesis of the preceding replicons in the sequence. This model predicts that DNA synthesis in mid or late S should depend upon the synthesis and/or integrity of previously replicating DNA, but should not depend upon the integrity of DNA replicating later in the sequence. By incorporating BUdR into DNA during a given short interval of one S period in synchronous Chinese hamster ovary cells, we are able to selectively damage this DNA by irradiation at selected times before or during the next S period. Utilizing this technique, we find that damage to early replicating DNA before entry into the second S phase markedly suppresses DNA synthesis in the entire S period. Damage to mid or late replicating DNA prior to entry into the second S period has no effect on early S, but markedly reduces DNA synthesis commencing in mid or late S, respectively. Furthermore, if early replicating DNA is damaged with light in mid-S, no effect on subsequent DNA synthesis is observed. These results can be fitted to a model in which sequential triggering of replicon synthesis promotes orderly progression through S.     

Archaeal histone tetramerization determines DNA affinity and the direction of DNA supercoiling

DNA binding and the topology of DNA have been determined in complexes formed by >20 archaeal histone variants and archaeal histone dimer fusions with residue replacements at sites responsible for histone fold dimer:dimer interactions. Almost all of these variants have decreased affinity for DNA. They have also lost the flexibility of the wild type archaeal histones to wrap DNA into a negative or positive supercoil depending on the salt environment; they wrap DNA into positive supercoils under all salt conditions. The histone folds of the archaeal histones, HMfA and HMfB, from Methanothermus fervidus are almost identical, but (HMfA)(2) and (HMfB)(2) homodimers assemble into tetramers with sequence-dependent differences in DNA affinity. By construction and mutagenesis of HMfA+HMfB and HMfB+HMfA histone dimer fusions, the structure formed at the histone dimer:dimer interface within an archaeal histone tetramer has been shown to determine this difference in DNA affinity. Therefore, by regulating the assembly of different archaeal histone dimers into tetramers that have different sequence affinities, the assembly of archaeal histone-DNA complexes could be localized and used to regulate gene expression.     

Maturation of the head of bacteriophage T4. III. DNA packaging into preformed heads

An estimate was made of the amount of DNA packaged into gene 49-defective heads when P49 is activated by a temperature shift. The uptake of DNA into preformed heads following activation of P49 was studied using bromo-deoxyuridine as a label. The rate of inactivation by visible light of the phage matured in the presence of BrdU as well as their buoyant density in CsCl, indicate that over half of the particles package, on the average, at least 25% of the DNA complement following P49 activation. This is a minimum estimate, since the BrdU-labeled DNA has to compete with unlabeled DNA. Analysis on alkaline sucrose gradients of the size of the DNA extracted from phage matured in the presence of BrdU following irradiation reveals that extended irradiation at 313 nm breaks the DNA close to half of its original size. These experiments clearly show that up to half of the DNA can be packaged into the preformed heads made at high temperature following activation of the product of gene 49 (P49), strongly supporting the pathway for phage head maturation described by Laemmli &; Favre (1973).The so-called τ-particles, which accumulate in 24-defective cells, can serve as precursors of the mature phage (Bijlenga et al., 1973). We have measured the uptake of BrdU-labeled DNA into τ-particles during their maturation. We find that a very large proportion of DNA made after activation of P24 is apparently incorporated into preformed τ-particles as these particles are converted into mature heads. This indicates that the τ-particles contain very little or no DNA prior to P24 activation and supports the pathway described by Laemmli &; Favre (1973).     

Plant mitochondria actively import DNA via the permeability transition pore complex

Plant mitochondria are remarkable with respect to their content in foreign, alien and plasmid-like DNA, raising the question of the transfer of this information into the organelles. We demonstrate the existence of an active, transmembrane potential-dependent mechanism of DNA uptake into plant mitochondria. The process is restricted to double-strand DNA, but has no obvious sequence specificity. It is most efficient with linear fragments up to a few kilobase pairs. When containing appropriate information, imported sequences are transcribed within the organelles. The uptake likely involves the voltage-dependent anion channel and the adenine nucleotide translocator, i.e. the core components of the mitochondrial permeability transition pore complex in animal cells, but it does not rely on known mitochondrial membrane permeabilization processes. We conclude that DNA import into plant mitochondria might represent a physiological phenomenon with some functional relevance.     

Insights into the replisome from the structure of a ternary complex of the DNA polymerase III alpha-subunit

The crystal structure of the catalytic α−subunit of the DNA polymerase III (PolIIIα) holoenzyme bound to primer-template DNA and an incoming deoxy-nucleoside 5′-triphosphate has been determined at 4.6-Å resolution. The polymerase interacts with the sugar-phosphate backbone of the DNA across its minor groove, which is made possible by significant movements of the thumb, finger, and β-binding domains relative to their orientations in the unliganded polymerase structure. Additionally, the DNA and incoming nucleotide are bound to the active site of PolIIIα nearly identically as they are in their complex with DNA polymerase β, thereby proving that the eubacterial replicating polymerase, but not the eukaryotic replicating polymerase, is homologous to DNA polymerase β. Finally, superimposing a recent structure of the clamp bound to DNA on this PolIIIα complex with DNA places a loop of the β-binding domain into the appropriate clamp cleft and supports a mechanism of polymerase switching.