Question

Question 30:

A. Describe the secondary structural characteristics of B-form double-stranded DNA.

B. Explain how to determine the number of supercoils present in a molecule of DNA.

C. Explain/demonstrate how to determine changes in linking number for DNA.

Answer 1

30

(A) DNA's secondary structure is predominantly determined by base-pairing of the two polynucleotide strands wrapped around each other to form a double helix.

B-form DNA

The information from the base composition of DNA, the knowledge of dinucleotide structure, and the insight that the X‑ray crystallography suggested a helical periodicity were combined by Watson and Crick in 1953 in their proposed model for a double helical structure for DNA. They proposed two strands of DNA -- each in a right‑hand helix -- wound around the same axis. The two strands are held together by H‑bonding between the bases (in anti conformation).

Bases fit in the double helical model if pyrimidine on one strand is always paired with purine on the other. From Chargaff's rules, the two strands will pair A with T and G with C. This pairs a keto base with an amino base, a purine with a pyrimidine. Two H‑bonds can form between A and T, and three can form between G and C. This third H-bond in the G:C base pair is between the additional exocyclic amino group on G and the C2 keto group on C. The pyrimidine C2 keto group is not involved in hydrogen bonding in the A:T base pair.

Dimensions of B-form (the most common) of DNA

• 0.34 nm between bp, 3.4 nm per turn, about 10 bp per turn
• 1.9 nm (about 2.0 nm or 20 Angstroms) in diameter

Major and minor groove

The major groove is wider than the minor groove in DNA and many sequence specific proteins interact in the major groove. The N7 and C6 groups of purines and the C4 and C5 groups of pyrimidines face into the major groove, thus they can make specific contacts with amino acids in DNA-binding proteins. Thus specific amino acids serve as H‑bond donors and acceptors to form H-bonds with specific nucleotides in the DNA. H‑bond donors and acceptors are also in the minor groove, and indeed some proteins bind specifically in the minor groove. Base pairs stack, with some rotation between them.

These are the complementary base pairs. The base‑pairing scheme immediately suggests a way to replicate and copy the the genetic information.

B). DNA supercoiling is important for DNA packaging within all cells. Because the length of DNA can be thousands of times that of a cell, packaging this genetic material into the cell or nucleus (in eukaryotes) is a difficult feat. Supercoiling of DNA reduces the space and allows for DNA to be packaged.

Unwinding of the helix during DNA replication (by the action of helicase) results in supercoiling of the DNA ahead of the replication fork.

• This supercoiling increases with the progression of the replication fork.
• If the supercoiling is not relieved, it will physically prevent the movement of helicase.

The "supercoil" is most likely a full 360°, rather than 180°. In any case, hold the ends of the duplex so that a left handed 360° "supercoil" is present.

The Writhe number refers to the number of supercoils present.

• Although it may seem that the consequence of introducing supercoiling (Writhe) is changing the Linking number, it is not.
• The consequence of Writhe is that the Twist (apparent linkage number) is altered (increased or decreased).

DNA has a preferred "Twist" value (preferred apparent linkage number) for a specified length of DNA:

• Watson and Crick's model of the DNA duplex had 10 basepairs per turn.
• Under physiological conditions of salt (0.15 M NaCl) and temperature, DNA prefers to adopt about 10.6 bp/turn.
• Writhe is introduced in the DNA to achieve this value for the "Twist" (apparent linkage number)

For a given (fixed) Linkage number over a given length of DNA, the DNA can adopt either positive or negative supercoils to achieve a "twist" (apparent linkage number) such that there will be 10.6 basepairs/turn.

C).

The topology of DNA can be described by three parameters:

1. Linking Number (L) An integer value. "Positive" is referenced as right-handed.
2. Twist (T) A real number (the "apparent" linkage number)
3. Writhe (W) A real number ("supercoils" in the DNA structure

Consider closed circular DNA:

• Linking number is an integer value.
• It refers to the number of times the two strands of the duplex make a complete 360 degree turn.

For circularly closed DNA, like the E. coli genome, the linking number can only be changed if we do the following:

1. physically break the duplex
2. introduce (or remove) a 360 degree turn
3. ligate (covalently close) the break.

Linkage number does not change with supercoiling (it can only change by breaking the duplex)

• Writhe has the effect of changing the apparent Linkage number.
• One supercoil is defined as being able to change the apparent linkage number by +/- 1.

The twist value (apparent linkage number) for a given length of DNA is related to the number of base pairs per turn that the DNA wants to adopt:

Linkage Number = (size of DNA in base pairs)/(basepairs/turn) + Writhe

or

Linkage Number = #of Twists + Writhe

this is usually abbreviated as

Linkage = Twist + Writhe

L = T + W