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Lecture 3 DNA Topology

DNA Topology

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Biomolecules

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Lecture 2 DNA topology

● DNA topology ○ Supercoiling ○ Twist ○ Writhe

Introduction ● Most bacteria have circular chromosomes

● Replication bubble and the replication fork ● This is actually a chromosome caught during replication. ● The two rabbit ears labelled A and C in the drawing are the new portion, the big one (B) is the unreplicated portion. ● The ‘armpits’ are the replication forks.

Plasmids are also circular, but much, much smaller

● This is the pSC101 plasmid, which was the first vector that was successfully used for molecular cloning

Lecture 2 DNA topology ● Actually, with 9263 bps it is much bigger than most of the modern vectors ○ E. The pUC18 vector is only 2686bp.

● Circular DNAs can have closed covalent or nicked forms

● RF1 is covalently closed and supercoiled but RFII is nicked - you can get multiple nicked one circle ● By convention, the closed covalent structure is called ‘replicative form’ I. It is usually supercoiled ● The single-nicked is RF II: this is the relaxed form ● On the agarose gel to the right, which band do you think is which? Why? ○ The supercoiled band is the bottom band.

● Two major bands

Using these circular molecules topology is easier to visualise

● Parameters that characterise DNA topology: twist, writhe, linking number ○ Twist/twisting number (Tw): ■ Number of times one strand crosses in front of the other strand - the helical turn - one twist is one helical turn

Lecture 2 DNA topology

● Relaxed DNA ● Has 25 twists ● 0 writhes ● Base pairs - 25x10.

● Force of twists is stronger than the force of writhes ● 10 base pairs per twists/helices ● Linking number always wants to be constant so whn writhe is introduced the twisting number will want to go back to 25 + the write is -2 so the linking number is 23.

● Numbering in the adapted version of the sample figure in Tropp is completely wrong.

Lecture 2 DNA topology

● Plasmids (small extra-chromosomal elements in bacterial cells) show different linkage numbers and super-helical state. ● 1 - relaxed DNA ● as we introduce writhes, the molecule is more compact ● Travel fast will go to the bottom of the gel so the supercoiled will be at the bottom as it is more dense because it is so coiled so it is heavier

Circular DNAs can form supercoils

● A double-stranded DNA molecule, shown for simplicity as a single line, is arranged in two different supercoiled forms. ● (a) - The DNA axis repeatedly crosses over and under itself (plectonemic supercoil). ● (b) - the DNA is arranged in a series of spirals around an imaginary ring (shown in magenta) to form a toroidal supercoil.

Lecture 2 DNA topology

Supercoiling in numbers ● Superhelical density:

● Supercoiling on top + linking number on the bottom ● That is, the ratio between supercoiling and the linking number of relaxed DNA ● It also describes the number of writhes per number of twists

● Can be presented as a fraction, decimal or a percentage

In most organisms, DNA is negatively supercoiled ● DNA is more organisms is negatively supercoiled ● The degree of underwinding in most organisms is 5%-7%; o = -0 to -0. ● Reverse calculating: ○ If o= -0, and Wr=1, then Tw = 20 ○ o= -0 and Wr = 1, then Tw = ~ ● Because the strain of twists is larger than the strain of writhes, the number of basepairs per twists is constant, ~10, the number of basepairs per writhe is 210-150 (if o is between -0 and -0) ● DNA of eukaryotes is linear, however, topological constraint also exists. Eukaryotic DNA is anchored to proteins, these chromatin domains are constrained topologically. ● Negative supercoiling stores torsional energy, which helps open up, unwind, the DNA for metabolism. ● Can you think of a scenario, where positive supercoiling, i. over-winding the DNA is advantageous? ○ To protect the DNA from becoming denatured in environments such as hot springs

Lecture 2 DNA topology

Topoisomerases catalyse strand passage

● Type I enzymes catalyse the passage of a single stranded molecule through a single- stranded gap ● Type II enzymes catalyse the passage of a double stranded molecule through a double- stranded gap

Lecture 2 DNA topology

DNA topoisomerase catalyses transient breakage of DNA

● Transesterification between an enzyme tyrosyl and a DNA phosphate group leads to the breakage of a DNA backbone bond and the formation of a covalent enzyme-DNA intermediate. ● Rejoining of the DNA backbone bond occurs by the reversal of the reaction shown. ● In the reaction that is catalysed by a type IA or a type II enzyme, a 3’ OH is the leaving group and the active-site tyrosyl becomes covalently linked to a 5’-phosphoryl group, as depicted ● In the reaction that is catalysed by a type IB enzyme (not shown), a 5’-OH is the leaving group and the active-site tyrosyl becomes covalently linked to a 3’-phosphoryl group

Lecture 2 DNA topology

● Reactions catalysed by eukaryotic topoisomerase II (TOP2) include decatenation of linked intact double stranded DNA and relaxation of supercoiled DNA. ● The reaction formally requires introduction of a double strand break, strand passage and break resealing. ● Topoisomerase II interacts with two DNA strands to effect strand passage. ● The enzyme introduces a double strand break in one DNA strand, termed the G or gate segment, and will pass a second strand termed the T segment through the break. ● In the presence of Mg2+, the enzyme can cleave the DNA, forming a phosphotyrosine linkage between each single strand and a tyrosine in each subunit. ATP binding causes the enzyme to form a closed clamp. ● The closed clamp may also capture another strand (the T strand) that will pass through the break made in the G strand. ● After passing through the break in the G strand, the T strand exits the enzyme through the carboxy terminus (the bottom of the enzyme as drawn). ● ATP hydrolysis occurs at two steps in the reaction cycle105. ● The first ATP hydrolysed may assist in strand passage. ● The second hydrolysis step (along with release of ADP and inorganic phosphate (Pi)) allows the clamp to reopen, and allows release of the G segment (for a distributive reaction). ● Alternatively, the enzyme may initiate another catalytic cycle without dissociating from the G strand.

Lecture 2 DNA topology

● Top1-mediated DNA relaxation by controlled rotation. ● By contrast to type IA or II enzymes, this reaction does not require an energy cofactor or divalent metal. ● Top1 tends to bind DNA crossovers (supercoils) and nicks DNA by transesterification (see Figure 1D). ● The enzyme then allows the DNA to swivel by controlled rotation. ● Upon DNA realignment by base pairing and stacking across the nick, the DNA 5′- hydroxyl end (OH in lower panels) removes the tyrosyl linkage by reverse transesterification (see Figure 1D). ● These enzymes form a covalent link with the 3’ end of the DNA at the nick.

Lecture 2 DNA topology

Summary ● DNA topology is very tightly controlled within the cells ● DNA metabolic processes require ‘opening up’ of DNA for access ● Maintaining the topological state of DNA inside cells is the job of topoisomerases ● Inhibition of topoisomerases has great therapeutic potential

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