Renaturation rate

The renaturation rate of DNA is an excellent indicator of the sequence complexity of DNA. For example, bacteriophage T4 DNA contains about 2 X 10 nucleotide pairs, whereas Escherichia coli DNA possesses 4.64 X 10 . E. coli DNA is considerably more complex in that it encodes more information. Expressed another way, for any given amount of DNA (in grams), the sequences represented in an E. coli sample are more heterogeneous, that is, more dissimilar from one another, than those in an equal weight of phage T4 DNA. Therefore, it will take the E. coli DNA strands longer to find their complementary partners and reanneal. This situation can be analyzed quantitatively. 

Gillis, M., J. De Ley, and M. De Cleene. 1970. The determination of molecular weight of bacterial genome DNA from renaturation rates. Europ. J. Biochem., 12 143-153. 

Thermal stability (decreased or increased hyperchromicity, higher or lower melting temperature and greater or lower renaturation rate, respectively)... 

COMPOUND AND TREATMENT HYPERCHROMICITY (% variation) ATm ( ) RENATURATION RATE (% variation)... 

To answer the question whether the ds-transisomerization of the bridged polypeptides with a Ala-Gly-Pro sequence represents the rate-determining step, the following experiment was carried out The polypeptide with a chain length n = 8 was denaturated in a rapid reaction with a temperature jump from 9.2 to 30 °C and subjected to renatura-tion at 9.2 °C after an incubation time of 25 s. In a second and a third experiment, the incubation in the coiled state was prolonged respectively to 75 and 125 s. It could be observed that the amplitude of the rapid phase depends on the time that lapses between the denaturation and renaturation (Fig. 32). 

One may conclude that the rate-determining step of the renaturation is at least partly influenced by the cis-trans isomerization of the peptide bond the secondary nitrogen atom of which arises from proline. Otherwise, only the entropy-controlled slow nuclea-tion should be observed kinetically. The covalent bridging through Lys-Lys, therefore, gives rise not only to thermodynamic stabilization of the triple helix but also to kinetic properties which have hitherto been observed in the case of type III procollagen146) and its aminoterminal fragment Col 1-3144). 

Electrochemistry of disulfide unit present in cytochrome c (cyt c) molecules on gold electrodes has also been reported [169]. Disulfide unit in cytochrome c is strongly adsorbed on Au electrodes and this slows down the electron-transfer rate to the heme group. More recently, Krylov et al. [170] have immobilized cytochrome c by self-assembling on the surface-modified Au electrodes. CV was applied to study how denaturation and renaturation of cytochrome c depend on the solution composition. 

Examining the Rate of Renaturation for Genomic DNA Isolated from E. Coli. 129... 

The rate of urea denaturation was inhibited by a variety of anions known to bind to the enzyme in decreasing order of effectiveness, pyrophosphate, 2 -CMP, phosphate, citrate, tartrate, and sulfate (353). This inhibition was greater at pH 5.6 than 7.3. The binding constants were the same as those estimated by inhibition of the enzymic reaction. As with the pH and temperature effects, the anions had no demonstrable effect on the rate of renaturation. 

Steps in denaturation and renaturation of a DNA duplex. In step l the temperature is raised to the point where the two strands of the duplex separate. If denatured DNA is slowly cooled, the events depicted as steps 2 and 3 follow. In step 2 a second-order reaction occurs in which two complementary strands of DNA must collide and form interstrand hydrogen bonds over a limited region. Step 3 is a first-order reaction in which additional hydrogen bonds form between the complementary strands that are partially hydrogen-bonded (zippering). Once complementary strands are partially bonded, the zippering reaction occurs rapidly. In the overall process, step 2 is rate-limiting. 

Kinetic analysis indicates that renaturation is a two-step process. In the slow step effective contact is made between two complementary regions of DNA originated from separate strands. This rate-limiting step called nucleation is a function of the concentration of complementary strands. Nucleation is followed by a relatively rapid zippering up of adjoining base residues into a duplex structure. The steps involved in denaturation and renaturation are depicted in figure 25.14. 

The rate of renaturation is also a function of chain length, but this effect is usually eliminated as a variable by shearing the starting DNA down to a uniform size. For a typical renaturation experiment the values of c/c0 are plotted as a function of cQt, and the resulting curve is referred to as a cot curve. 

In mammals, some 10% of the DNA is highly repetitive, a further 20% is moderately reiterated and the remainder represents non-repetitive or single copy sequences. The rate of renaturation of DNA has been used to estimate that 16% of the DNA in H. diminuta is repetitive (746). Furthermore, the rate of reassociation of the single copy component in this species has allowed calculation of its haploid genome size, which is approximately... 

Fig. 3.4 Temperature-dependent reconstitution of tetrameric K coli aspartase.29 A Reactivation of denatured aspartase. The enzyme denatured in 4 M guanidine-HCl was renatured at 4° C by dilution. After 14 min, the temperature of each preparation was shifted up as indicated in the figure. The temperature of each preparation was further shifted up to 30° C after 45 min. B HPLC analysis of intermediates in the renaturation process. Aspartase renatured at 4°C was incubated for 15 min at the indicated temperatures. An aliquot of each preparation was applied to a TSKgel G3000SWXL column (7.5 X 300 mm) and eluted with a flow rate of 0.5 ml/ min. The temperature of the sample in the sample loop, elution buffer and the column was maintained constant. (From Physiol Chem. Phys. Med. NMR, 21, 222 226 (1989)).

Denatured DNA can be renatured the separated DNA strands can be annealed under proper circumstances, and the rate of annealing has provided valuable information on DNA structure. Annealing of native DNA strands is often incomplete, and for this reason, the denatured DNA may be broken into smaller fragments by shearing. Sheared DNA anneals completely. Annealing is a... 

The rate of renaturation depends on the concentration of complementary sequences. Viral DNA has a smaller variety of sequences than does bacterial DNA this reflects the higher level of genetic complexity in bacteria. Thus, for viral and bacterial DNA fragments of the same average size and at the same molar concentration, there would be a higher concentration of complementary sequences in the former. Viral DNA therefore would renature faster than bacterial DNA. In other words, bacterial DNA has greater sequence heterogeneity. 

Rates of renaturation and sequence heterogeneity (or complexity) can be examined quantitatively through COT analysis. If Q is the initial concentration of DNA (moles per liter DNA phosphate) and k is the rate constant for association of the complementary strands, it can be shown that the fraction / of single-stranded molecules decreases with time t (s) according to the expression... 

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