Topology Flashcards
Why are topoisomerase’s required?
- To relive the positive supercoiling force generated by helicase as the DNA strands separate
How do topoisomerase’s relive the strain on the DNA strands? (Brief)
By pumping negative supercoils into replicating DNA
How is superhelicity maintained in plasmid and viral DNA?
Through the covalently closed circular nature of the duplex
How are supercoiled loops in bacterial and Eukaryotic chromosomes restrained?
Through the protein scaffold of the chromosome
Define Linking number (4)
- In relaxed duplex L = # of Watson Crick turns
- # of times one strand crosses over the other
- L = sum of twist and writhe of duplex
- L must be an integer
What is twist?
Number of helical turns in the DNA
What is writhe?
Number of superhelical turns in the DNA double helix
How does Ethidium Bromide effect DNA in Vitro? (3)
- Allows visualisation of DNA as acts a base so binds to the DNA.
- Introduces +ve supercoiling into DNA
- Too high conc it makes DNA brittle.
What are the main features of TOPO 1? (5)
- Increase L number by 1
- Nick and pass mechanism
- No ATP required
- Doesn’t resolve supercoiling ahead of rep fork
- Relaxes -ve supercoils only
What are the main features of TOPO 2?
- Decrease L number by 2
- Cut and pass mechanism
- ATP required
- Introduces -ve supercoiling into DNA
- Relaxes +ve supercoiling
Why does TOPO 1 not require ATP but TOPO 2 does?
Due to the energy for the breakage being conserved in the Tyr-P intermediate formed. TOPO 2 cuts 2x so needs to reset the enzyme. ATP is required for this.
What is the TOP10 mutation?
- Nonesene mutation
- Results in an increase in -ve supercoiling as mutation in top1 gene so topoisomerase can’t relax -ve supercoiling
What is the TOP- mutation?
- No Topo1 activity
- Less supercoiling than top10 mutation
- Never occurs in isolation
- Can cause normal supercoiling as type 1 defective but type 2 can introduce -ve supercoiling into DNA
