lecture 21 - DNA/RNA structure cont.

how is DNA compacted

supercoiling

relaxed DNA vs supercoiled DNA

what kind of DNA can be supercoiled

• supercoiling can only exist in a DNA molecule where both strands of DNA are closed circles or fixed at one end.

supercoiling: what happens if one strand breaks

if one strand breaks, the DNA rapidly loses its supercoiling (relaxes)

how is the topology(shape) of dsDNA defined?

by linking number

what is linking number

number of times one strand would have to be passed through the other strand in order for the strands to be separated from each other

two components: Lk = Tw(twist) + Wr(writhe)

ALWAYS AN INTEGER

CANNOT BE CHANGED unless one or both strands of the duplex is broken

Tw, twist

• for dsDNA, twist is number of full turns of the helix, for B form helix, bp = 10.5, ex. for a segment 105 bp long twist = 105/10.5 = 10

Wr, writhe

• degree of supercoiling

• number of times double helix crosses itself

• left handed ds helix:

  • wr is positive

  • overwinding (+) makes it harder to separate strands of ds

  • BRLO (b form, left handed, overwinding)

• right handed ds helix:

  • wr is negative

  • underwinding (-) makeas it easier to separate strands of ds

  • BRRU (b form, right handed, underwinding)

• wr for relaxed dsDNA is 0

example of relationship between Lk = Tw + Wr

• strain of local disruption of base pairing in double helix (c ) may be taken up by supercoiling in (b) to restore Tw to 36.

• Linking number DOES NOT CHANGE

what level of structure is supercoiling

tertiary

supercoiling & gel electrophoresis

• a supercoiled DNA molecule is more compact than a relaxed DNA molecule of the same length, it moves faster than relaxed DNA in gel electrophoresis

• use ethidium bromide (EthBr)

• DNA gel electrophoresis separates DNA fragments based on their size and shapes

• in general, smaller & more compact DNA fragments move quicker

what are topoisomers

• DNA molecules in different coiled forms that have the same nucleotide sequence

• change superhelicity

what are topoisomerases

• enzymes that catalyze topoisomers (change superhelicity)

what do topoisomeraes do and how(general process)?

topoisomeraes change linking number of DNA by catalyzing a three step process:

1. cleave one or both strands of dsDNA

2. the passage of a segment of DNA through the break

3. resealing of the DNA breaks

topoisomeraes have a key tyrosine residue in their active site that covalently attaches to a phosphate in the sugar-phosphate backbone that is transiently broken

other roles of topoisomerase

• they help relieve torsional stress in replication, transcription, and recombination for both prokaryotes & eukaryotes, by unwinding the DNA during these events

types of topoisomerases & what they do

The degree of supercoiling of DNA is maintained by regulation of the net activity of topoisomerase types I and II.

type I

type II

nicks one strand of DNA, the other strand then passes through the ss break, and the broken strand re-seals, adding or removing one turn at a time changes Lk by increments of 1 thermodynamically favorable process driven by release of energy of supercoiling - releases the strain of supercoiled DNA E. coli type I topoisomerases generally relax DNA by removing negative supercoils (increasing Lk)

breaks both strands, then re-seals both strands, adding or removing 2 writhes at a time changes Lk in increments of 2 can relax negative and positive supercoils, thermodynamically favourable in E. coli, type II topoisomerase (DNA gyrase) can also decrease the linking number to introduce negative supercoils – this process is not thermodynamically favorable- requires energy input – type IIuses energy from ATP hydrolysis

topoisomers & agarose gel

• A population of mostly supercoiled circular DNA molecules shown in Lane 1 has been treated with Topo I in Lanes 2 and 3 (for a longer period of timein Lane 3).

• The products of Topo I treatment migrate to different points in the gel depending on their linking number, Lk; each band differs by a single Lk (i.e., these DNA molecules are topoisomers).

• With long enough Topo I treatment, all of the supercoiled DNA will be completely relaxed.

why are topoisomerases the target of antibacterial and anti-cancer drugs

• without topoisomerases, cells cannot replicate or package their DNA, so they die

• quinolones(bad) inhibit a type II topoisomerase by blocking the last step of the topoisomerase rxn, resealing of the DNA strand breaks. this inhibits the unwinding of DNA, preventing cell division