Rate of renaturation

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. 

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... 

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... 

Hutton, J.R., and J.G. Wetmur. 1973. Effect of chemical modification on the rate of renaturation of deoxyribonucleic acid deamination and glyoxalated deoxyribonucleic acid. Biochemistry 12 558-563. 

Fig. 1. The relationship between the kinetics of renaturation of DNA, in terms of CQti ( ) or 1/k2(o), and genome size (in daitons). There is an essentially linear decrease in the rate of renaturation with increase in genome size. (From the data of Britten and Kohne. 1968. Sc/eA7ce, 161 529-540 and Wetmur and Davidson. 1968. Molec.Biol.,3 349-370.)...

Fig. 2. The kinetics of reassociation of calf thymus DNA measured with hydroxyapatite. The DNA was sheared to small fragments and the single-stranded fragments incubated under renaturing conditions. At various times samples were passed over a hydroxyapatite column at 60 C, under conditions in which only duplex DNA binds to hydroxyapatite. Because of the large range in Cot values, different initial concentrations (Cq) of calf DNA were used to obtain the complete renaturation curve A, 2 jug/nni , 10/xg/ml o, 600Mg/ml and 8,600 /ug/ml. The crosses are radioactively labeled E. coli DNA at 43 jug/ml present in the renaturing mixture of calf thymus DNA at 8,600 The rate of renaturation of E. coli DNA provides an internal standard with which the...

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. 

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... 

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