Lecture 21 Flashcards
1
Q
What is DNA superhelicity
A
- a way of compacting DNA
2
Q
How do cells contain/package/handle their DNA
A
- bacterial DNA can be compacted by a process called supercoiling into a nucleoid
- DNA is condensed, organized and segregated with the help of topoisomerase enzymes, nucleoid associated proteins and the SMC complex
- eukaryotic DNA is packaged in chromatin
3
Q
Where can supercoiling exist
A
- only in a DNA molecule where both strands of DNA are closed circles or otherwise fixed at one end
- if one strand breaks the DNA rapidly loses its supercoiling
4
Q
What are topoisomers
A
DNA molecules in different coiled forms that have the same nucleotide sequence
5
Q
What does untwisting DNA helix at one position do
A
- changes the superhelicity
- pulling apart the strands of a helix will induce over-winding, or supercoiling in the other end
- the ends must either be covalently closed (as in ccc dsDNA), or topologically constrained (as in eukaryotes, by scaffolding proteins)
6
Q
What is the linking number
A
- can be used to define the topology (shape) of dsDNA
- is the number of times one strand would have to be passed through the other strand in order for the strands to be completely separated from each other
- sum of two geometric components
- Lk = Tw + Wr
7
Q
What is Tw (twist)
A
- geometrical property of the helix
- for dsDNA twist is the number of full turns of the helix
- Tw = # of bp/10.5
8
Q
What is Wr (writhe)
A
- the number of times the double helix crosses itself
- if ds helix writhes in the left-handed directions, Wr is (+) - overwinding makes it more difficult to separate the strands of the double helix
- if helix writes in the right-handed direction, Wr is (-) - underwinding makes it easier to separate the strands of the double helix
9
Q
What can cause the Lk to change
A
- if one or both of the strands of the duplex is broken
- supercoiling markedly alters the overall form of DNA
- relaxed DNA can lie flat on a planar surface
- since a supercoiled molecule has extra twists in its helix axis itself, it cannot lie flat
- supercoiled DNA represents higher order folding of secondary, or helix, and is called the tertiary structure
- a supercoiled DNA molecule is more compact than a relaxed DNA molecule of the same length, it moves faster than relaxed DNA when subjected to centrifugation or gel electrophoresis
10
Q
DNA gel electrophoresis
A
- migration depends on the degree of supercoiling
- because of the negatively-charged phosphates in the sugar-phosphate backbone, DNA is (-) charged and will migrate to the anode (+) in a buffered agarose gel
- smaller DNA fragments move through the gel more quickly than larger
- more compact, supercoiled DNA fragments more faster through the gen than less supercoiled fragments of the same size
- the DNA bands can be visualized under UV light by staining with ethidium bromide - dye that binds the DNA by intercalating between the bases
11
Q
What is agarose
A
- polysaccharide polymer, extracted from seaweed
- slabs of agarose gel are prepared by dissolving agarose in gel buffer, microwaving this solution, and then pouring it out into a mold to set
12
Q
What type of relationship is it between the log of DNA size and the distance
A
- linear relationship
13
Q
What do topoisomerases do
A
- change the linking number of DNA by catalyzing a 3 step process
1. the cleavage of one or both strands of dsDNA
2. the passage of a segment of DNA through this break
3. resealing of the DNA breaks - play important roles in replication, transcription, and recombination for both prokaryotes and eukaryotes - help relieve torsional stress induced by unwinding the DNA during these events
- have a key tyrosine residue in their active site that covalently attaches to a phosphate in the sugar-phosphate backbone that is transiently broken
14
Q
Explain type 1 topoisomerases
A
- 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 the linking number by increments of 1
- thermodynamically favorable process driven by release of energy of supercoiling- releases the strain of supercoiled DNA
- E. coli type 1 topoisomerases generally relax DNA by removing negative supercoils (increasing Lk)
15
Q
Explain type 2 (DNA gyrase in bacteria) topoisomerases
A
- breaks both strands, and then reseals both strands, adding or removing 2 writhes at a time
- changes Lk in increments of 2
- can relax negative supercoils and positive supercoils (thermodynamically favorable processes)
- in E. coli, type 2 topoisomerase (DNA gyrase) can also decrease the linking number to introduce negative supercoils (not thermodynamically favorable) - requires energy input
- type 2 uses energy from ATP hydrolysis
