The adenovirus DNA binding protein enhances intermolecular DNA renaturation but inhibits intramolecular DNA renaturation.

The Adenovirus DNA binding protein (DBP) imposes a regular, rigid and extended conformation on single stranded DNA (ssDNA) and removes secondary structure. Here we show that DBP promotes renaturation of complementary single DNA strands. Enhancement of intermolecular renaturation is sequence independent, can be observed over a broad range of ionic conditions and occurs only when the DNA strands are completely covered with DBP. When one strand of DNA is covered with DBP and its complementary strand with T4 gene 32 protein, renaturation is still enhanced compared to protein-free DNA, indicating that the structures of both protein-DNA complexes are compatible for renaturation. In contrast to promoting intermolecular renaturation, DBP strongly inhibits intramolecular renaturation required for the formation of a panhandle from an ssDNA molecule with an inverted terminal repeat. We explain this by the rigidity of an ssDNA-DBP complex. These results will be discussed in view of the crystal structure of DBP that has recently been determined.     

Renaturation of complementary DNA strands by herpes simplex virus type 1 ICP8.

ICP8, the major single-stranded DNA-binding protein of herpes simplex virus type 1, promotes renaturation of complementary single strands of DNA. This reaction is ATP independent but requires Mg2+. The activity is maximal at pH 7.6 and 80 mM NaCl. The major product of the reaction is double-stranded DNA, and no evidence of large DNA networks is seen. The reaction occurs at subsaturating concentrations of ICP8 but reaches maximal levels with saturating concentrations of ICP8. Finally, the renaturation reaction is second order with respect to DNA concentration. The ability of ICP8 to promote the renaturation of complementary single strands suggests a role for ICP8 in the high level of recombination seen in cells infected with herpes simplex virus type 1.     

Excluded volume effects on the rate of renaturation of DNA

The rate of renaturation of T2 DNA hits been investigated by using complementary DNA strands of different length. The length of the shorter strand ranged from 0.02 to 1.0 times the length of the longer strand. An excluded volume theory is developed to include this type of reaction as well as the DNA–RNA hybridization reaction. Experimental and theoretical rates of renaturation of DNA are found to be in agreement. For the cases studied, the rate was never greater than twice that observed for short strands of the same length renaturing with themselves. The products of renaturation reactions are also considered.     

Mechanism of DNA denaturation in the presence of manganese ions

The unwinding of DNA strands in the presence of small concentrations of Mn²⁺ ions (2 × 10^?4?4 × 10^?4M) has been studied. The process of unwinding is nonequilibrium; the DNA strands are gradually unwound at a constant temperature corresponding to the beginning of the melting curve. There is no true renaturation in the partially melted DNA. It is shown in the paper that these effects are due to the aggregation of the unwound DNA regions. The Mn²⁺ ions are responsible for the binding of the unwound strands. The aggregation precludes renaturation, shifts the equilibrium towards the melted state, and causes slow unwinding at a constant temperature. The binding of denaturated regions seems to occur through the guanines.     

Kinetic modeling of the recA protein promoted renaturation of complementary DNA strands

Quantitative agarose gel assays reveal that the recA protein promoted renaturation of complementary DNA strands (phi X DNA) proceeds in two stages. The first stage results in the formation of unit-length duplex DNA as well as a distribution of other products ("initial products"). In the second stage, the initial products are converted to complex multipaired DNA structures ("network DNA"). In the presence of ATP, the initial products are formed within 2 min and are then rapidly converted to network DNA. In the absence of ATP, the initial products are formed nearly as fast as with ATP present, but they are converted to network DNA at a much lower rate. The time-dependent formation of initial products and network DNA from complementary single strands for both the ATP-stimulated and ATP-independent reactions can be modeled by using a simple two-step sequential kinetic scheme. This model indicates that the primary effect of ATP in the recA protein promoted renaturation reaction is not on the initial pairing step (which leads to the formation of initial products) but rather is to increase the rate at which subsequent pairing events can occur.     

The homologous recombination system of phage lambda. Pairing activities of beta protein

The red genes of phage lambda specify two proteins, exonuclease and beta protein, which are essential for its general genetic recombination in recA- cells. These proteins seem to occur in vivo as an equimolar complex. In addition, beta protein forms a complex with another polypeptide, probably of phage origin, of Mr 70,000. The 70-kDa protein appears to be neither a precursor nor an aggregated form of either exonuclease or beta protein, since antibodies directed against the latter two proteins failed to react with 70-kDa protein on Ouchterlony double diffusion analysis. beta protein promotes Mg2+-dependent renaturation of complementary strands (Kmiec, E., and Holloman, W. K. (1981) J. Biol. Chem. 256, 12636-12639). To look for other pairing activities of beta protein, we developed methods of purification to free it of associated exonuclease. Exonuclease-free beta protein appeared unable to cause the pairing of a single strand with duplex DNA; however, like Escherichia coli single strand binding protein (SSB), beta protein stimulated formation of joint molecules by recA protein from linear duplex DNA and homologous circular single strands. Like recA protein, but unlike SSB, beta protein promoted the joining of the complementary single-stranded ends of phage lambda DNA. beta protein specifically protected single-stranded DNA from digestion by pancreatic DNase. The half-time for renaturation catalyzed by beta protein was independent of DNA concentration, unlike renaturation promoted by SSB and spontaneous renaturation, which are second order reactions. Thus, beta protein resembles recA protein in its ability to bring single-stranded DNA molecules together and resembles SSB in its ability to reduce secondary structure in single-stranded DNA.     

DNA conformational kinetics

A model for the time dependence of DNA conformational state probabilities is formulated in the form of first-order differential equations. This model is applied to investigate the renaturation and denaturation rates for T2 and T7 DNA as reported in the series of experiments by Record and Zimm. Qualitative agreement is found in denaturation and for series of renaturation experiments with the same initial condition. However, partial agreement with series of renaturation experiments having the same final condition is obtained only by including an initial bimolecular step with properly matched pairs of strands. Comparison of all experiments with the calculated rates yields 5 × 10⁴ min^?1 as the step rate for melting a single base pair.     

Rapid assembly and disassembly of complementary DNA strands through an equilibrium intermediate state mediated by A1 hnRNP protein.

A1 hnRNP protein, which rapidly renatures complementary strands of nucleic acids in vitro, affects both the equilibrium and kinetic properties of the reaction (single-stranded DNA in equilibrium with double-stranded DNA). A1 lowers the melting transition of duplex DNA. However, at temperatures above this new Tm, both single- and double-stranded DNAs are present at equilibrium and are rapidly interconverting. Although the ratio of single and double strands under these conditions is a function of both the A1 protein and complementary DNA strand concentrations, it is not strongly affected by further increases in temperature. These surprising results demonstrate that A1 does not act as a simple catalyst in promoting renaturation and indicate how A1 and other proteins could act to speed the turnover of intermediate complexes in important biological processes.     

Renaturation of DNA: a novel reaction of histones.

Histones isolated from several sources, either singly or in combination promote the renaturation of complementary single strands of DNA, as measured by the acquisition of resistance to S1 nuclease. The reaction is rapid (T1/2 less than 1 min), and is stoichiometric rather than catalytic. Renaturation is stimulated by Mg2+, Mn2+, and Ca2+, but is strongly inhibited by Zn2+. Crude extracts of early embryos of Drosophila melanogaster possess renaturation activity which is protease sensitive, heat-stable, and acid-soluble, suggesting that most or all of it can be attributed to histones. This observation thus provides a functional assay for histones that should prove useful in studies of chromatin and histone-DNA interactions, as well as for the identification and isolation of histones and histone-like proteins in crude extracts.     

Naturally occurring cross-links in yeast chromosomal DNA.

Chromosome-size yeast DNA molecules with a number average molecular weight (Mn) of 3-4 X 10(8) were isolated from sucrose gradients after sedimentation of lysed yeast spheroplasts. Resedimentation showed that the molecules were isolated without introducing appreciable single-strand or double-strand breaks. The presence of cross-links in these molecules was suggested by the observation that the apparent Mn in alkali was greater than expected for separated single strands. Since cross-linked molecules would have strands which fail to separate upon denaturation, this was tested more directly. Neutralization of alkaline denaturing conditions resulted in up to 70% of the intact molecules rapidly reforming duplex structures, as shown by equilibrium banding in CsCI. Experiments with larger E. coli DNA molecules (Mn = 5.2 X 10(8)) indicated that the conditions used were sufficient to denature completely molecules of this size. Results of enzyme treatments suggest that the cross-links are not RNA or protein. Experiments with density-labeled yeast DNA molecules showed that the rapid reformation of duplex DNA is not the consequence either of a bimolecular reaction between separated DNA strands or of intrastrand renaturation. The data indicate that when the yeast DNA molecules are completely denatured, the strands fail to separate. Hence they must be cross-linked. Experiments with sheared DNA show that there are small number of cross-links, one to four, permolecule.     

The Renaturation of Denatured DNA

The kinetics of renaturation of heat-denatured DNA from E. coli and pneumococcus have been examined by ultraviolet absorption measurements. The molecularity of the reaction was assessed by three independent treatments of the data, and all lead to the conclusion that renaturation is essentially first order at 60°; at 70° and 80° there is an increasing second order component, resulting in simultaneous unimolecular and bimolecular kinetics. The unimolecular kinetics rule out reaction between two, kinetically separate strands, indicating rather the zippering-up of a single, denatured entity. The bimolecular kinetics can be attributed to the complexing of two such entities; thus, the genetic or density-labeled complexes that have been observed by other investigators can be accounted for without invoking strand separation. Since renaturation at best is never complete, the free ends of two renatured molecules permit sufficient bimolecular reaction to produce density hybrids. The observed kinetics can be accounted for if the hydrogen bonds of DNA are broken during heat denaturation but the strands do not separate. Light scattering supports this by showing that the molecular weight is unchanged by denaturation. Since there is no existing evidence that is inconsistent with this hypothesis, it is reasonable to conclude that heat denaturation does not completely separate the entangled strands of the DNA molecule.     

Intrastrand self-complementary sequences in Bacillus subtilis DNA.

Intrastrand self-complementary sequences have been isolated from the DNA of Bacillus subtilis by hydroxyapatite (HA) chromatography following thermal renaturation of strands separated by chromatography on methylated albumin kieselguhr (MAK). The instrastrand structures derived from the MAK H strand (HA HII) were biologically active showing transforming activity for a wide variety of markers, as well as hybridization to both pulse-labelled and ribosomal RNA. Removal of regions of single-strand DNA with S1 nuclease did not significantly alter the biological activity of the self-annealed molecules. The overall efficiency of transformation and hybridization of the intrastrand self-annealing DNA was low suggesting that many sequences in the population are neither active in transformation to prototrophy nor transcribed into RNA.     

Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: Acceleration of the renaturation rate

The thermal stability and renaturation kinetics of DNA have been studied as a function of dimethyl sulfoxide (DMSO) concentration. Increasing the concentration of DMSO lowers the melting temperature of DNA but results in an increased second-order renaturation rate. For example, in a DNA solution containing 0.20M NaCl, 0.01M Tris (pH 8.0), and 0.001M EDTA, the addition of 40% DMSO lowers the melting temperature of the DNA by 27°C and approximately doubles the optimal renaturation rate. The effect of DMSO on the renaturation rate is shown to be at least partially due to its effect on the solution dielectric constant and to be consistent with the polyelectrolyte counterion condensation theory of Manning [(1976) Biopolymers 15 , 1333–1343].     

ATP-independent renaturation of complementary DNA strands by the mutant recA1 protein from Escherichia coli

In an effort to clarify the requirement for ATP in the recA protein-promoted renaturation of complementary DNA strands, we have analyzed the mutant recA1 protein which lacks single-stranded DNA-dependent ATPase activity at pH 7.5. Like the wild type, the recA1 protein binds to single-stranded DNA with a stoichiometry of one monomer per approximately four nucleotides. However, unlike the wild type, the mutant protein is dissociated from single-stranded DNA in the presence of ATP or ADP. The ATP analogue adenosine 5'-O-3' (thiotriphosphate) appears to stabilize the binding of recA1 protein to single-stranded DNA but does not elicit the stoichiometry of 1 monomer/8 nucleotides or the formation of highly condensed protein-DNA networks that are characteristic of the wild type recA protein in the presence of this analogue. The recA1 protein does not catalyze DNA renaturation in the presence of ATP, consistent with the dissociation of recA1 protein from single-stranded DNA under these conditions. However, it does promote a pattern of Mg2+-dependent renaturation identical to that found for wild type recA protein.     

DNA strand exchange activity of rice recombinase OsDmc1 monitored by fluorescence resonance energy transfer and the role of ATP hydrolysis

Rad51 and disrupted meiotic cDNA1 (Dmc1) are the two eukaryotic DNA recombinases that participate in homology search and strand exchange reactions during homologous recombination mediated DNA repair. Rad51 expresses in both mitotic and meiotic tissues whereas Dmc1 is confined to meiosis. DNA binding and pairing activities of Oryza sativa disrupted meiotic cDNA1 (OsDmc1) from rice have been reported earlier. In the present study, DNA renaturation and strand exchange activities of OsDmc1 have been studied, in real time and without the steps of deproteinization, using fluorescence resonance energy transfer (FRET). The extent as well as the rate of renaturation is the highest in conditions that contain ATP, but significantly less when ATP is replaced by slowly hydrolysable analogues of ATP, namely adenosine 5'-(beta,gamma-imido) triphosphate (AMP-PNP) or adenosine 5'-O-(3-thio triphosphate) (ATP-gamma-S), where the former was substantially poorer than the latter in facilitating the renaturation function. FRET assay results also revealed OsDmc1 protein concentration dependent strand exchange function, where the activity was the fastest in the presence of ATP, whereas in the absence of a nucleotide cofactor it was several fold ( approximately 15-fold) slower. Interestingly, strand exchange, in reactions where ATP was replaced with AMP-PNP or ATP-gamma-S, was somewhat slower than that of even minus nucleotide cofactor control. Notwithstanding the slow rates, the reactions with no nucleotide cofactor or with ATP-analogues did reach the same steady state level as seen in ATP reaction. FRET changes were unaffected by the steps of deproteinization following OsDmc1 reaction, suggesting that the assay results reflected stable events involving exchanges of homologous DNA strands. All these results, put together, suggest that OsDmc1 catalyses homologous renaturation as well as strand exchange events where ATP hydrolysis seems to critically decide the rates of the reaction system. These studies open up new facets of a plant recombinase function in relation to the role of ATP hydrolysis.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 2. DNA renaturation reaction.

We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA). In the absence of nucleotide cofactor, the recA protein first converts the complementary single strands into unit-length duplex DNA and other relatively small paired DNA species; these initial products are then slowly converted into more complex multipaired network DNA products. ATP and PTP stimulate the conversion of initial product DNA into network DNA, whereas ITP and GTP completely suppress network DNA formation. The formation of network DNA is also inhibited by all four of the corresponding nucleoside diphosphates, ADP, PDP, IDP, and GDP. Those nucleotides which stimulate the formation of network DNA are found to enhance the formation of large recA-ssDNA aggregates, whereas those which inhibit network DNA formation cause the dissociation of these nucleoprotein aggregates. These results not only implicate the nucleoprotein aggregates as intermediates in the formation of network DNA, but also establish the functional equivalency of ITP and GTP with the nucleoside diphosphates. Additional experiments indicate that the net effect of ITP and GTP on the DNA renaturation reaction is dominated by the corresponding nucleoside diphosphates, IDP and GDP, that are generated by the NTP hydrolysis activity of the recA protein.     

A study of the reversibility of helix-coil transition in DNA.

The reversibility of DNA melting has been thoroughly investigated at different ionic strengths. We concentrated on those stages of the process that do not involve a complete separation of the strands of the double helix. The differential melting curves of pBR 322 DNA and a fragment of T7 phage DNA in a buffer containing 0.02M Na+ have been shown to differ substantially from the differential curves of renaturation. Electron-microscopic mapping of pBR 322 DNA at different degrees of unwinding (by a previously elaborated technique) has shown that the irreversibility of melting under real experimental conditions is connected with the stage of forming new helical regions during renaturation. In a buffer containing 0.2M Na+ the melting curves of the DNAs used (pBR322, a fragment of T7 phage DNA, a fragment of phage Lambda DNA, a fragment of phiX174 phage DNA) coincide with the renaturation curves, i.e. the process is equilibrium. We have carried out a semi-quantitative analysis of the emergence of irreversibility in the melting of a double helix. The problem of comparing theoretical and experimental melting curves is discussed.     

Kinetics of renaturation of denatured DNA. I. Spectrophotometric results

The kinetics of renaturation of heat- or formamide-denatured DNA have been studied by following the change of optical density at a constant temperature. Solvents of different ionic strength and various DNA samples have been used. At the lower ionic strengths studied, the reaction follows second-order kinetics, substantiating the hypothesis that strands of native DNA separate upon denaturation and recombine during renaturation. As the ionic strength is increased at a constant temperature, the reaction deviates from simple second-order behavior. This appears to be the result of the inhibition to rewinding caused by short helical segments in the denatured DNA which are more stable at the higher ionic strenth.