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Name 4 different ways that DSBs arise

- Nuclease driven
- VDJ recombination
- Meiotic recombination


What uses nuclease induced DSBs and why?

Used by Yeast for mating type switching


Describe the process of mate switching in Yeast

1. HO endonuclease makes a DSB of 24 bases on chromosome 3 on MATalpha locus

2. This locus uses info from silent loci on either side of it (HMLalpha and HMRa) to repair break to switch mating type to MATa


What are the two different mating types in yeast?

- MATalpha
- MATa


How can we repair nuclease induced DSBs?



What does the CRISPR/Cas9 system allow us to do?

It is a mechanism that allows us to make a specific DSB in mammalian cells to allow aberrant repairs leading to deletions/mutations so that we can study the phenotype.


Describe how we can use the CRISPR/Cas9 system

1. Piece of viral DNA incorporated into CRISPR locus

2. This will make a DSB in a target sequence at specific locus

3. = CRISPR guided RNA

2. Cas9 nuclease will cut at that specific target locus via the CRISPR guided RNA


What is a con of using the CRISPR/Cas9 system?

- Although it is made to make a specific DSB at specific locus based on expression of viral DNA

- Still risk of off-target effect = small deletions and mutations at off-target sites


What is VDJ recombination so important?

Because it creates antibody diversity during immune development


What does VDJ recombination result in?

Creates DSBs within immunoglobulin genes


What makes the DSBs in VDJ recombination?

RAG1 and RAG2 nucleases


Describe the VDJ recombination pathway using RAG1/2

1. RAG1/2 cleaves the pairing complex in VDJ recombination = a pair of hairpins

2. Artemis opens the hairpins

3. The ends are then ligated together via NHEJ pathway creating new joins


Describe the meiotic recombination pathway

1. DSB created during pro-phase by SPO11 enzyme (which is an ancestral topoisomerase)

2. Stays covalently attached to the 5' ends of the DNA break

= this stops inappropriate repair so repair can be directed by a specific way by HR

= also no repair can happen until these stupid enzymes are removed


What is meiotic recombination for?

- For genetic diversity in haploid gametes

- Facilitates reductional chromosome segregation during nuclear division


What is the difference between DNA repair in mitosis and meiosis?

Mitosis: NHEJ during replication or HR if its after replication. HR done between sister chromatids.

Meiosis: HR between non sister chromatids


What is the role of it topoisomerase?

Deals with catenation and supercoiling


What is catenation?

When either 2 DNA molecules are linked together and can't separate or there is one big DNA molecule that has lots of loops that are intertwined with each other i.e. has lots of catenations.


When do cantenations happen?

Arises due to semi conservative replication
(chromosomes condense and then tries to segregate)


Why do we need topoisomerases?

Needed to resolve catenations by making transient DNA breaks which then will be ligated by a repair pathway

(Also needed for DNA replication and segregation)


Describe the topoisomerase 2 pathway

1. Topo 2 will bind to the gate strand

2. A clamp will capture a transfer strand

3. Topo will cleave the G strand and make a link between 5' DSB ends of the G strand and a catalytic tyrosine in each topo subunit

4. T strand will then pass through the break made in the G strand

5. T strand exits through C terminus and the broken G strand is resealed


How can we fix topoisomerase induced breaks?

- If break appears before S phase: NHEJ

- If break appears after S phase: HR


How are natural nuclease driven (yeast) DSBs repaired?



How are engineered nuclease driven (CRISPR) DSBs repaired?

Either NHEJ or HR


How are VDJ recombination DSBs repaired?



How are meiotic recombination DSBs repaired?



What causes non physiological DSBs?

- Ionising radiation: gamma/X-rays make 20 SSBs per DSB.

- Oxidative damage: free radicals make 200 SSBs per DSB

- Error in DNA replication

- Error in topoisomerase activity: failure to reseal DSB


What is the difference between the roles of topo 1 and topo 2?

- Topo1 cuts single strands

- Topo 2 cuts double strands


How can we fix DSBs caused by failed topo 2?

1. Topo2 forms dimer

2. Some sort of DNA damage will happen

3. Topo dimer attaches onto DNA to make a DSB which is usually reversible

4. But denaturation proteolysis may break up the topo leading bits stuck to the 5' ends of the DSB, this is an irreversible DSB

5. So then TDP2 enzyme will remove those broken bits of topo by phosphorylation

6. Now the DNA strands can be ligated back together via NHEJ or HR


Why do cancer cells need topoisomeration?

For proliferation


What is etoposide and how does it work?

- It is an anti-cancer drug.

- It stalls topo2 on DNA until it either denatures or some sort of DNA machinery comes along to push it off

- So if there isn't any topo activity, DNA replication can't happen because the DNA strand is all twisted and shit