lec20 Flashcards
how do bacteria cells contain/package their dna
Bacterial DNA is compacted by supercoiling into a nucleoid, aided by topoisomerases, nucleoid-associated proteins, and the SMC (structureal maintenance of chrom)complex
– topoisomerase: “i just got some top from a stripper bitch” and then king von like gets all tight from the head
What enzymes and proteins help organize bacterial DNA?
Topoisomerases (regulate supercoiling), nucleoid-associated proteins (assist in organization), and the SMC complex (helps in chromosome maintenance and segregation).
Supercoiling can’t exist in a DNA molecule where both strands of DNA are closed
circles or otherwise fixed at one end. t or f
f
Supercoiling can only** exist in a DNA molecule where both strands of DNA are closed
circles or otherwise fixed at one end.
DNA molecules in different coiled forms that have the same nucleotide sequence
are called ________
topoisomers
What is Tw (Twist) in DNA?
Twist (Tw) refers to the number of complete turns in the DNA helix.
In relaxed DNA, one full turn of the helix equals about 10.5 base pairs in B-form DNA (sometimes simplified to 10 base pairs).
The twist is a measure of how tightly the DNA is coiled.
What is Wr (Writhe) in DNA?
Writhe (Wr) refers to how much the DNA helix twists around itself.
Positive writhe (Wr +) happens when the DNA twists in the left-handed direction (overwinding).
Negative writhe (Wr -) happens when the DNA twists in the right-handed direction (underwinding).
In relaxed DNA, Wr = 0, meaning no extra twisting around itself.
Wr helps DNA manage strain when it is coiled or untangled.
overwinded is harder to untwist/seperate strands of thje double helix than underwinded (right)
How does DNA handle strain from local disruption of base pairing, and what happens to the linking number (Lk)? (ss in notes mar13)
When DNA experiences strain (e.g., base pairs are disrupted), it can take up this strain by supercoiling (twisting around itself).
This restores the twist (Tw) back to a normal level, such as Tw = 36.
The linking number (Lk), which represents the total number of twists, remains unchanged even when DNA supercoils. The supercoiling just redistributes the strain without changing the overall twists in the DNA.
What is the difference between relaxed and supercoiled DNA, and how does supercoiling affect DNA?
Relaxed DNA: Untwisted, can lie flat on a planar surface without extra twists in the helix axis.
Supercoiled DNA: Has extra twists in its helix axis, making it more compact and unable to lie flat. It represents the tertiary structure of DNA and moves faster during centrifugation or gel electrophoresis. Supercoiling alters the overall shape of DNA and is a higher-order folding of its secondary structure.
What is the linking number (Lk) in circular DNA, and how can it be changed?
Lk (Linking Number) is a topological property of circular DNA.
It represents the total number of twists in the DNA.
Lk cannot be changed unless one or both of the strands of the DNA are broken.
It is a constant value for a given circular DNA unless the strands are physically cut.
Why does DNA migrate toward the anode in gel electrophoresis, and how do DNA fragment sizes and supercoiling affect migration?
DNA is negatively charged because of the negatively charged phosphates in its sugar-phosphate backbone, so it migrates towards the anode (+) in an agarose gel.
Smaller DNA fragments move faster through the gel than larger fragments.
More compact, supercoiled DNA fragments move faster than less supercoiled fragments of the same size.
DNA bands can be visualized under UV light after staining with ethidium bromide, which binds to DNA by intercalating between the bases.
How does the percentage of agarose gel affect the migration of DNA fragments?
Higher percentage agarose gel: Creates smaller pores, slowing down larger DNA fragments. Use this for smaller DNA fragments so they can separate better.
Lower percentage agarose gel: Creates larger pores, allowing larger DNA fragments to move more easily. Use this for larger DNA fragments.
what is on the axis for linear graph of dna gel electrophoresis
log of dna size and distance migrated gives linear graph
What do topoisomerases do, and how do they change the linking number of DNA?
Topoisomerases are enzymes that change the shape (supercoiling) of DNA by altering its linking number.
They do this in a three-step process:
1. Cleaving one or both strands of the DNA.
2. Passing a segment of DNA through the break.
3. Resealing the DNA breaks.
This helps to relieve torsional stress caused by DNA unwinding during processes like replication, transcription, and recombination.
How does topoisomerase work at the molecular level, and what is its role in DNA processes?
Topoisomerases have a special tyrosine residue in their active site that attaches covalently to a phosphate in the DNA sugar-phosphate backbone.
This bond temporarily breaks the DNA, allowing the enzyme to relieve supercoiling.
Topoisomerases are crucial for packaging DNA in cells and are involved in replication, transcription, and recombination for both prokaryotes and eukaryotes.
type i and ii topoisomerases
Topoisomerases Flashcard
Type I Topoisomerase
Cuts one DNA strand; the other strand passes through the break.
Changes Lk by ±1 per step.
Relaxes negative supercoils (increases Lk).
No ATP needed (strain energy drives process).
Example: E. coli Topo I.
Type II Topoisomerase (e.g., DNA Gyrase in Bacteria)
Cuts both DNA strands, then reseals them.
Changes Lk by ±2 per step.
Can relax negative & positive supercoils (thermodynamically favorable).
DNA gyrase introduces negative supercoils (decreases Lk, requires ATP not thermodynamically favoured).
Regulation
DNA supercoiling is controlled by the balance of Type I and Type II topoisomerase activity.
What effect does supercoiling have on DNA migration in an agarose gel?
What happens to DNA after treatment with Topo I?
Supercoiled DNA is more compact and moves faster and farther down the gel.
Topo I treatment relaxes the DNA by removing negative supercoils (increasing Lk). Relaxed DNA moves slower in the gel due to decreased compactness.
Topoisomers formed by different degrees of relaxation will migrate to different points in the gel, with each band differing by one Lk.
How do Topo I and Topo II differ in their effects on DNA supercoiling?
Topo I only relaxes supercoils (removes negative or positive supercoils).
Topo II (like DNA gyrase in bacteria) can both relax and introduce supercoils.
dna gyrase
topo II in e. coli
can also decrease Lk to introduce negative supercoils (with atp, not favoured thermdynam)
Introducing negative supercoils requires ATP and helps keep bacterial DNA underwound, making replication and transcription easier.
Positive supercoils naturally form ahead of replication and transcription, so bacteria counteract this by actively adding negative supercoils.
drugs and topoisomerases
Bacterial drugs: Quinolones (e.g., Ciprofloxacin) inhibit DNA gyrase (Topo II) by blocking DNA resealing, preventing replication and cell division.
Cancer drugs: Target Topo I or Topo II, which are elevated in tumor cells, making them more toxic to cancer cells than normal cells.
