Lecture 21 Flashcards
What is DNA superhelicity
- a way of compacting DNA
How do cells contain/package/handle their DNA
- 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
Where can supercoiling exist
- 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
What are topoisomers
DNA molecules in different coiled forms that have the same nucleotide sequence
What does untwisting DNA helix at one position do
- 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)
What is the linking number
- 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
What is Tw (twist)
- geometrical property of the helix
- for dsDNA twist is the number of full turns of the helix
- Tw = # of bp/10.5
What is Wr (writhe)
- 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
What can cause the Lk to change
- 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
DNA gel electrophoresis
- 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
What is agarose
- 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
What type of relationship is it between the log of DNA size and the distance
- linear relationship
What do topoisomerases do
- 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
Explain type 1 topoisomerases
- 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)
Explain type 2 (DNA gyrase in bacteria) topoisomerases
- 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
How is the degree of supercoiling of DNA maintained
- the degree of supercoiling of DNA is maintained by regulation of the net activity of topoisomerase types 1 and 2
What happens with a longer Topo 1 treatment
- all of the supercoiled DNA will be completely relaxed
Topoisomerases are the molecular targets of antibacterial and anti-cancer drugs
-Cellular DNA function relies heavily on its topological state, managed by topoisomerases.
-Topoisomerases are essential for DNA replication, packaging, gene expression, and cell viability.
-Inhibitors of topoisomerases, such as quinolones for bacteria and specific agents for cancer, are effective in treating infections and malignant cells respectively.
