Location of the cooperative melting regions in bacteriophage fd DNA

Differential melting profiles of the linear replicative form (RF-III) DNA of bacteriophage fd, of the fragments obtained by the restriction endonuclease R.HinHI and of those obtained by R.Hga were investigated. With these results a physical map which locates the cooperative melting regions on the DNA was constructed, and compared with the genetic map.     

The effect of sequence heterogeneity on DNA melting kinetics

We consider kinetics of the cooperative melting of DNA sections situated at the edge of the helix. Accurate calculations based on the real sequences of such sections demonstrate that their internal heterogeneity has a drastic effect on the melting kinetics. Allowance for the internal heterogeneity increases the relaxation time by several orders of magnitude as compared with a model based on the assumption of equal base-pair stability within a section. The relaxation times obtained are in good agreement with the experimental data of Suyama and Wada (A. Suyama and A. Wada, Biopolymers, 23, 409 (1984)). An analysis of the melting process revealed some simple sequence characteristics that determine its rate. An examination of the temperature dependence of the relaxation time led to a distinct interpretation of the apparent activation energies of the denaturation and renaturation. The relaxation time proved to reach its maximum near the equilibrium melting point of the section examined.     

Distribution of charges in Bacillus intermedius 7P ribonuclease determines the number of cooperatively melting regions of the globule

A correlation between the distribution of charged side groups in the globule of Bacillus intermedius 7P ribonuclease (binase) and the process of heat denaturation was studied at different pH values in order to estimate a relation between charge distribution in globular proteins and the character of cooperative thermodynamic transitions. As was shown by comparing the results of scanning microcalorimetric analysis of heat denaturation with the three-dimensional structure of binase, at optimal pH the molecule exists as a single cooperative system stabilized by hydrogen bonds, Van der Waals' contacts, and electrostatic interactions like salt bridges. At pH lower than 4.0 (below the physiological optimum) the cooperativity type of the system was found to change due to a reversible cooperative transition in the ternary structure of the protein globule. It has been concluded that the molecular architecture and the arrangement of atoms do not change considerably in different environments; thus the thermodynamic properties of the globule vary due to the alteration of charge distribution and the consequent changes in the size and number of cooperative regions of the globule. Thus, structural and energetic domains may be non-coincident in proteins.     

Preparative isolation and study of the reassociation kinetics of a T2 phage DNA fraction containing readily melting regions]

A DNA fraction comprising 6% of total DNA and containing readily-melting regions is isolated from phage T2 DNA using preparative chromatography on MAK columns at T congruent to T m--3 degrees C. Two denaturation regions, differing in the stability for 7 degrees, were observed on this DNA melting curve. A sharp increase of the reassociation rate at initial moments under reassociation temperatures T r approximately less than m --25 degrees C was observed. Thermodynamic characteristics obtained under the repeated melting of DNA fragments after reassociation confirm the fact, that under these reassociation temperatures the incorporation of readily-melting regions into spiral duplexes takes place.     

Unwinding of forked DNA structures by UvrD

Many studies have demonstrated the need for processing of blocked replication forks to underpin genome duplication. UvrD helicase in Escherichia coli has been implicated in the processing of damaged replication forks, or the recombination intermediates formed from damaged forks. Here we show that UvrD can unwind forked DNA structures, in part due to the ability of UvrD to initiate unwinding from discontinuities within the phosphodiester backbone of DNA. UvrD does therefore have the capacity to target DNA intermediates of replication and recombination. Such an activity resulted in unwinding of what would be the parental duplex DNA ahead of either a stalled replication fork or a D-loop formed by recombination. However, UvrD had a substrate preference for fork structures having a nascent lagging strand at the branch point but no leading strand. Furthermore, at such structures the polarity of UvrD altered so that unwinding of the lagging strand predominated. This reaction is reminiscent of the PriC-Rep pathway of replication restart, suggesting that UvrD and Rep may have at least partially redundant functions.     

Unwinding of unnatural substrates by a DNA helicase

Helicases separate double-stranded DNA into single-stranded DNA intermediates that are required during replication and recombination. These enzymes are believed to transduce free energy available from ATPase activity to unwind the duplex and translocate along the nucleic acid lattice. The nature of enzyme-substrate interactions between helicases and duplex DNA substrates has not been well-defined. Most helicases require a single-stranded DNA overhang adjacent to duplex DNA in order to initiate unwinding. The strand containing the overhang is referred to as the loading strand whereas the complementary strand is referred to as the displaced strand. We have investigated the interactions between a DNA helicase and the DNA substrate by replacing the displaced strand with a nucleic acid mimic, peptide nucleic acid (PNA). PNA is capable of forming duplex structures with DNA according to Watson-Crick base pairing rules, but contains a N-(2-aminoethyl)glycine backbone in place of the deoxyribose phosphates. The PNA-DNA hybrids had higher melting temperatures than their DNA-DNA counterparts. Dda helicase, from bacteriophage T4, was able to unwind the DNA-PNA substrates at similar rates as DNA-DNA substrates. The results indicate that the rate-limiting step for unwinding is relatively insensitive to the chemical nature of the displaced strand and the thermal stability of oligonucleotide substrates.     

Statistics of RNA melting kinetics

We present and study the behavior of a simple kinetic model for the melting of RNA secondary structures, given that those structures are known. The model is then used as a map that. assigns structure dependent overall rate constants of melting (or refolding) to a sequence. This induces a landscape of reaction rates, or activation energies, over the space of sequences with fixed length. We study the distribution and the correlation structure of these activation energies. Correspondence to: P. Schuster     

Unwinding of DNA induced by fluorescein mercuric acetate

Fluorescein mercuric acetate causes the unwinding of DNA and binds to the separated bases. This unwinding process can be followed by measuring absorption changes of this reagent. For untreated calf thymus DNA, the initial rate was very slow, and the shape of the kinetic curve was sigmoidal. When double-strand breaks of DNA were produced by DNase II treatment or sonication, the initial rate increased and the sigmoidal character disappeared. The initial rate was shown to be proportional to the concentration of helix ends. From this relation the rate of unwinding was estimated to be 2.0 base pairs/sec at 1.0 × 10^?5M fluorescein mercuric acetate and 25°C. DNase I treatment, which produces single-strand breaks and a smaller number of double-strand breaks, also increased the initial rate. However, this increase was due only to the double-strand breaks, that is, single-strand breaks had no significant effect on the initial rate. Also, uv irradiation increased the initial rate linearly with uv dose, at least up to 2 × 10⁵ erg/mm², suggesting that this increase is due to photoproducts other than usual pyrimidine dimers. We discuss the usefulness of this kinetic method in structural studies of DNA.     

Letter: Unwinding the DNA helix by intercalation

Data relating to the effect of intercalating drugs on the winding of the DNA helix is re-considered. Analyses by Paoletti &; Le Pecq (1971) of fluorescence depolarization, X-ray diffraction and molecular model building are reappraised. It is concluded that the helix is unwound by ~12 ° as proposed by Fuller &; Waring (1964). This refutes the recent suggestion by Paoletti &; Le Pecq (1971) that the intercalation winds the helix by ~13 °.     

Structural characteristics (reassociation and melting kinetics) of kinetoplast DNA from Crithidia oncopelti

A highly purified associate of kinetoplast DNA is isolated from C. oncopelti, and its physico-chemical properties are studied. Both native associate and its ultrasonic fragments are found to have a complex character of melting. 5-6 melting zones (3 of them being the main) are found on the melting curve. Analysis of reassociation kinetics of sonicated associate of kinetoplast DNA has revealed the presence of at least two components: fast reassociating component (65-70% of complex DNA), which reassociation kinetics is equivalent to the unique sequence with molecular weight of 2.3. - 10(6) daltons, and slow reassotiating component (15% of complex DNA), having reassociation kinetics equivalent to unique sequence of 26 - 10(6) daltons. The data obtained suggest that complex associate of kinetoplast DNA is heterogenous for its nucleotide sequence and base composition.     

A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase

Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ^−/− cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome.     

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding

The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation.     

The relation of single-stranded regions in bacteriophage PM2 supercoiled DNA to the early melting sequences.

Bacteriophage PM2 supercoiled DNA contains one to three small single-stranded regions that can be detected in the electron microscope after various treatments. The relative positions of these regions were mapped against the unique cleavage site for the restriction endonuclease R · HapII on PM2 DNA. Any of eight sharply defined regions of the genome may be single-stranded in supercoiled molecules. They are found in all possible combinations of three or less and at approximately the same frequency. A comparison of this map of supercoiled DNA with the alkaline denaturation pattern of nicked circular or linear PM2 DNA showed that these same regions were also the earliest melting regions in non-supercoiled DNA.     

DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity

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