DNA Supercoiling Lecture Notes PDF

Correction to lecture 3 On slide 7 L3, I posed the question of why the B-DNA double helix was 20 Å in diameter where as the distance between 1’ carbons of the opposite strands is ~11 Å One student said it was because the deoxyriboses add distance and I said that...

Correction to lecture 3 On slide 7 L3, I posed the question of why the B-DNA double helix was 20 Å in diameter where as the distance between 1’ carbons of the opposite strands is ~11 Å One student said it was because the deoxyriboses add distance and I said that was not correct…BUT SHE WAS PARTIALLY RIGHT!!! The diameter of the helix is wider than the 1’ carbon distances b/w two strand because of: i) the additional size of the deoxyribose and the phosphates ii) because the helix itself is is slightly wider still (see the ‘hole’ in the middle of the B-DNA structure below on the right?) ~11Å < 20Å 20.4Å 1 L4. Polynucleotide structure (2) - Supercoiling 2  DNA supercoiling In general, a coiled string can form supercoil when it is overt twisted or under twisted. See a phone cord shown to the right, note the coil and the supercoil. The phone cord is a coiled string, it forms supercoil if over (or under) twisted. DNA is like a coiled string, it can form supercoil when it is over twisted or under twisted. Atomic force microscopy image of supercoiled plasmid DNA from Lyubchenko and Shlyakhtenko (2016)3  info : to compact genetic purpace - Positive and negative supercoiling DNA double helix has natural helical turns (coils). Like a coiled string, it can be ‘relaxed’ or ‘supercoiled’. DNA forms supercoils if it is over twisted or under twisted Over twist is done in same direction as the natural helical turns in B-DNA (right-handed direction), making the DNA 'overwound’. Overwound DNA à forms positive supercoils. Under twist is done in opposite direction of the natural helical turns in DNA, making the DNA ‘underwound’. Underwound DNA à forms negative supercoils. 4  Supercoiling of circular DNA The circular relaxed DNA (shown to the right) has no supercoil. Relaxed DNA: one helical turn per 10.5 bp DNA BUT if the circular relaxed DNA is overwound (made to have more helical turns), it forms positive supercoil. If the circular relaxed DNA is underwound (made to have less helical turns), it forms negative supercoil. For DNA supercoiling to occur, both strands of the DNA must be intact. Cannot have a break (nick) in either DNA strand. ↳ if nicks, mraves, super col arand mension n to relieve Panels from left to right: relaxed DNA to highly supercoiled circular DNA (electron micrographs) 5  Linear vs. circular chromosomes and supercoiling In most Bacteria (and Archaea) the genome is comprised of a circular DNA chromosome that is supercoiled In eukaryotes, the nuclear genome consists of linear DNA chromosomes Ø Linear DNA can form supercoils when it is between two E. coli supercoiled circular chromosome & anchored points. A chromosome formed from linear DNA is pictured on the left In the expanded view, the linear DNA (colored blue) is attached to the chromosome scaffold (colored yellow, proteins) at many constrained points (anchored points). ① ⑨ Linear DNA between two constrained points can be relaxed DNA, but can also form supercoils if overwound or underwound ·= anchoring points 6 in the context of B-DNA One helical turn = 10.5bp Calculating the number of supercoils Lk: Linking number (times one strand passes over the other) Ø Actual number of helical turns in the DNA molecules you are considering 10.5 bp Lko: Linking number in totally relaxed DNA with the same number of base pairs Lko = number of base pairs divided by 10.5 S: Number of supercoils. (S is sometimes denoted W for ‘writhe’) S = Lk – Lko When S is a negative number (0), DNA has positive supercoil(s). For example, when S = -6, the DNA has 6 negative supercoils. 7  How many bp in the DNA? Lko = 200 Lko = 200 helical turns 200 x 10.5 bp/turn = 2,100 bp On the left S = Lk – Lko = 198 – 200 = -2 > - I & S = -2 has 2 negative supercoils & ( + 2(() + 1 & - On the right S = Lk – Lko = 202 – 200 = 2 S=2 has 2 positive supercoils 8 In most cells, DNA usually is negatively supercoiled Ø that is, the DNA is underwound. Right picture shows a section of a circular DNA. (a) The relaxed DNA has 8 helical turns. (b) The strained DNA is underwound by one helical turn. (c) To accommodate the strain, the DNA will negatively supercoil The DNA supercoiling is spontaneous, because it relieves structural stress of the strained DNA. 9 In most cells, DNA usually is negatively supercoiled Ø that is, the DNA is underwound. Right picture shows a section of a circular DNA. (a) The relaxed DNA has 8 helical turns. (b) The strained DNA is underwound by one helical turn. (c) To accommodate the strain, the DNA will negatively supercoil The DNA supercoiling in (c) is spontaneous, because it relieves structural strain of the underwound DNA in (b). An alternative way to accommodate the strain in (b) is to have strand separation (d) The strand separation is equivalent to adding a helical turn, thus changing the negatively supercoiled DNA to a relaxed DNA without a supercoil. un pull strands rep. apart (like ferk) Cellular DNA usually is negatively supercoiled (underwound) Easier for DNA strand separation. Easier for starting DNA replication and transcription. 10 6  DNA replication and transcription involve DNA strand separation induced supercoils (a) For a 2-stranded twisted rope, strand separation causes the rope to be overwound and form positive supercoils. overward underward (b) Transcription, direction of transcription to the right. + for RNA RNA not good The “fork” pushes the helical turns to the right. transcription i have enzyme to relieve tension in In front of the moving fork, DNA becomes overwound supercoils - forms positive supercoils. Behind the moving fork, DNA becomes underwound forms negative supercoils. For transcription to continue, need topoisomerases to relieve the overwound and underwound stress 2 11 memorize (Relaxes positive supercoils) unlink circles ↳ 2 circular chromosomes (bacterial ; Need to memorize the topoisomerases in bacteria only. Topo I and Topo II act in opposition, maintain appropriate levels of supercoiling. Topo III and Topo IV functions will be discussed in future lectures. Next lecture we will discuss Type I and Type II topoisomerases in detail 12