Topoisomerases and DNA Gyrase

The total amount of twisting present in a DNA molecule is referred to as the linking number (L). This is the sum of the contributions due to the double helix plus the super-coiling. [The number of double helical turns is sometimes known as the twist, T, and the number of superhelical turns as the writhe or writhing number, W. In this terminology, the linking number, L, is the sum of the twist plus the writhe (L = T + W).]

covalently closed circular DNA (cccDNA) Circular DNA with no nicks in either strand linking number (L) The sum of the superhelical turns (the writhe, W) plus the double helical turns (the twist, T)

open circle Circular DNA with one strand nicked and hence with no supercoiling twist, T The number of double helical turns in a molecule of DNA (or double-stranded RNA)

writhe Same as writhing number, W

writhing number, W The number of supercoils in a molecule of DNA (or double-stranded RNA)

FIGURE 4.17 Mechanism of Type I and II Topoisomerases

The difference in action between topoisomerases of Type I and Type II is in the breakage of strands. Type I breaks only one strand, while Type II breaks both strands. When one strand is broken, the other strand is passed through the break to undo one supercoil. When two strands are broken, double stranded DNA is passed through the break and the supercoiling is reduced by two. After uncoiling, the breaks are rejoined.

Supercoiled DNA

Supercoiled DNA

Type I

topoisomerases ->

one strand broken

Type I

topoisomerases ->

one strand broken

One strand passed through nick and resealed >

One strand passed through nick and resealed >

Type II

both strands broken

Type II

both strands broken

Both strands passed through cut and resealed

Both strands passed through cut and resealed

Enzymes known as topoisomerases change the level of supercoiling.

Ciprofloxacin kills bacteria by inhibiting DNA gyrase. It is harmless to animals as they do not use DNA gyrase for compacting their DNA.

The same circular DNA molecule can have different numbers of supercoils. These forms are known as topological isomers, or topoisomers. The enzymes that insert or remove supercoils are therefore named topoisomerases. Type I topoisomerases break only one strand of DNA, which changes the linking number in steps of one. In contrast, type II topoisomerases (including DNA gyrases) break both strands of the DNA and pass another part of the double helix through the gap. This changes the linking number in steps of two (Fig. 4.17).

DNA gyrase, a type II topoisomerase, introduces negative supercoils into closed circular molecules of DNA, such as plasmids or the bacterial chromosome. Gyrase works by cutting both strands of the DNA, introducing a supertwist and rejoining the DNA strands. Gyrase can generate 1,000 supercoils per minute. As each supertwist is introduced, gyrase changes conformation to an inactive form. Reactivation requires energy, provided by breakdown of ATP. DNA gyrase can also remove negative supercoils (but not positive ones) without using ATP, but this occurs ten times more slowly.

DNA gyrase is a tetramer of two different subunits. The GyrA subunit cuts and rejoins the DNA and the GyrB subunit is responsible for providing energy by ATP hydrolysis. DNA gyrase is inhibited by quinolone antibiotics, such as nalidixic acid and their fluorinated derivatives such as norfloxacin and ciprofloxacin, which bind to the GyrA protein. An inactive complex is formed in which GyrA protein is inserted into the DNA double helix and covalently attached to the 5'-ends of both broken DNA strands. Novobiocin also inhibits gyrase by binding to the GyrB protein and preventing it from binding ATP.

ciprofloxacin A fluoroquinolone antibiotic that inhibits DNA gyrase

DNA gyrase An enzyme that introduces negative supercoils into DNA, a member of the type II topoisomerase family nalidixic acid A quinolone antibiotic that inhibits DNA gyrase norfloxacin A fluoroquinolone antibiotic that inhibits DNA gyrase novobiocin An antibiotic that inhibits type II topoisomerases, especially DNA gyrase, by binding to the B-subunit quinolone antibiotics A family of antibiotics, including nalidixic acid, norfloxacin and ciprofloxacin, that inhibit DNA gyrase and other type II

topoisomerases by binding to the A-subunit topoisomerase Enzyme that alters the level of supercoiling or catenation of DNA (i.e. changes the topological conformation) topoisomers Isomeric forms that differ in topology—i.e. their level of supercoiling or catenation type I topoisomerase Topoisomerase that cuts a single strand of DNA and therefore changes the linking number by one type II topoisomerase Topoisomerase that cuts both strands of DNA and therefore changes the linking number by two

Supercoiling Affects DNA Structure 91

FIGURE 4.18 Unlinking of Catenanes by Topoisomerase IV

Topoisomerases may uncoil, unknot or unlink DNA as well as carrying out the coiling, knotting or interlinking of DNA. Topoisomerases act (at the locations shaded blue) by cutting both strands of the DNA at one location and passing another region of the DNA through the gap.

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