Relaxed DNA

Fig. 4. Laboratory tubing model for supercoiling in closed-circular DNA (a) relaxed DNA, ALK = 0 (b) ALK = ATw -...

DNA duplex of 400 bp, L is 40 (assuming 10 bp per turn in B-DNA). The linking number for relaxed DNA is usually taken as the reference parameter and is written as Lq. L can be equated to the twist (T) and writhe (W) of the duplex, where twist is the number of helical turns and writhe is the number of supercoils ... 

The chromosomes of Escherichia coli and other bacteria are single, double-stranded DNA molecules with a total length of more than 1,000 pm. Relaxed DNA exists as a helical molecule, with one full turn of the helix occurring approximately every 10.4 base pairs. This molecule must undergo several folding and compaction steps to fit into an E. coli cell which is only 1-3 pm long. Despite this enormous compaction, bacterial DNA must be accessible for the bacterial enzymes that catalize DNA replication and transcription... 

We can now describe DNA underwinding in terms of changes in the linking number. The linking number in relaxed DNA, Lk0, is used as a reference. For the molecule shown in Figure 24-16a, Lk(j = 200 if two turns are removed from this molecule, Lk = 198. The change can be described by the equation... 

It is often convenient to express the change in linking number in terms of a quantity that is independent of the length of the DNA molecule. This quantity, called the specific linking difference (cr), or superhelical density, is a measure of the number of turns removed relative to the number present in relaxed DNA ... 

FIGURE 24-17 Negative and positive supercoils. For the relaxed DNA molecule of Figure 24-16a, underwinding or overwinding by two helical turns (Lk = 198 or 202) will produce negative or positive supercoiling, respectively. Note that the DNA axis twists in opposite directions in the two cases. 

DNA supercoiling is a precisely regulated process that influences many aspects of DNA metabolism. Every cell has enzymes with the sole function of underwinding and/or relaxing DNA. The enzymes that increase or decrease the extent of DNA underwinding are topoisomerases the property of DNA that they change is the linking number. These enzymes play an especially im-... 

FIGURE 24-19 Promotion of cruciform structures by DNA under-winding. In principle, cruciforms can form at palindromic sequences (see Fig. 8-21), but they seldom occur in relaxed DNA because the linear DNA accommodates more paired bases than does the cruciform structure. Underwinding of the DNA facilitates the partial strand separation needed to promote cruciform formation at appropriate sequences. 

DNAs that differ only in linking number are called topoisomers. Enzymes that underwind and/or relax DNA, the topoisomerases, catalyze changes in linking number. The two classes of topoisomerases, type I and type II, change Lk in increments of 1 or 2, respectively, per catalytic event. 

The energy source for the process is provided by cleavage of ATP. DNA gyrase untwists relaxed circular dsDNA one turn at a time and reseals the cut ends. A reverse gyrase from certain bacteria twists the relaxed DNA more tightly. In both cases the change causes the DNA to form superhelical turns.67193-197 These may be either solenoidal or plectonemically interwound (as a twisted thread Fig. 5-18). 

Gyrase Type II topoisomerase that catalyzes conversion of relaxed DNA to a superhelical form requires ATP ... 

The reaction mixture contained 50 mM Tris-HCl buffer (pH 7.5), 120 mM KC1,10 mM MgCfe, 0.5 mM EDTA, 0.5 mM dithiothreitol, 30 /xg/mL bovine serum albumin, and 0.2 fig of pBR329 DNA. After incubation at 37°C for 30 minutes, the reaction was terminated by the addition of Sarkosyl in a final concentration of 1%. The reaction mixture was further incubated at 37°C for 30 minutes after proteinase K had been added to give a concentration of 50 /xg/mL. Assays were linear with up to 0.4 fig of topoisomerase I, and with time until about 95% of the substrate had been converted into relaxed DNA. 

Figure 9.152 Profile of separation of relaxed DNA from supercoiled plasmid pBR329 on a DEAE-NPR column. The reaction was carried out with 1 unit of calf thymus topoisomerase I. Twenty microliters of the reaction mixture was applied on the column and eluted with a linear gradient of 0.5 to 0.65 M NaCl for 30 minutes. DNAs from peaks a and b were collected manually and analyzed by electrophoresis on a 1% agarose gel after ethanol precipitation. Inset O.C., open circular S.C., supercoiled. (From Onishi et al., 1993.)...

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