Lecture 43. Recombination and Transposons

Question 1

What is recombination?

Answer 1

The breaking and rejoining of DNA into a new combination

Question 2

Why is recombination important?

Answer 2

Creates genetic diversity
“Easy” to do in eukaryotic cells which are diploid
In bacteria requires fragments of DNA from transformation, transduction and conjugation

Question 3

What is homologous recombination?

Answer 3

Switching DNA that is similar e.g same gene different allele

Question 4

What is non-homologous recombination?

Answer 4

Repair of double stranded DNA break by simply joining with another piece of DNA

Question 5

What does homologous recombination require?

Answer 5

Extensive homology (similarity of sequence)

Question 6

What can homologous recombination result in?

Answer 6

Faulty gene

Question 7

Homologous recombination mechanism 1

Answer 7

Alignment - 2 homologous DNA helices align
Breakage - On strand is nicked, E. Coli enzyme RecBCD, often happens at specific sequences
Invasion - Free 3’ end invades the homologous helix, DNA stabilised by SSB protein, catalysed by RecA

Question 8

What does RecA do and where is it found?

Answer 8

Has functional homologous in ALL known organisms incl. eukaryotes and archaea
Essential for DNA repair
Multiple function: binds single stranded DNA, has two binding sites so can hold two pieces of DNA together, catalyses branch mitigation

Question 9

What does RecBCD do?

Answer 9

Has nuclease activity and catalyses initial nick in DNA needed for recombination – note requires specific DNA sequence so this is not a random event
Has helicase activity – unwinds DNA after nick so single stranded DNA-binding protein and RecA can bind

Question 10

Homologous recombination mechanism 2

Answer 10

Cross strand exchange - A second nick in the other piece of DNA
Branch migration - Requires RuvAB helicase, extensive heteroduplexes form can be 1000s of bp long

Question 11

What is isomerisation?

Answer 11

Crossing and uncrossing of strands

Question 12

How many outcomes can a Holliday junction result in?

Answer 12

2

Question 13

What can rescue a double strand break?

Answer 13

Homologous recombination

Question 14

What are examples of non-homologous recombination?

Answer 14

Insertion and excursion of λ phage
Transposon events
Double stranded break repair

Question 15

Non-homologous recombination after DS breaks

Answer 15

After double strand break, the ends are either rejoined or joined to the “wrong” ends. If ends have been degraded then sequence may be lost

Question 16

What are insertion sequences?

Answer 16

Small pieces (~1000 bp) of DNA that can hop from one position to another

Question 17

What is the hop that an insertion sequence called and what catalyses it?

Answer 17

The hop is called transposition and is catalysed by transposase

Question 18

Insertion sequence phenotype consequences

Answer 18

Genes can be disrupted due to insertion but there is a high degree of reversion as insertion sequence simply moves somewhere else. If housekeeping gene disrupted, it can be fatal

Question 19

What are transposons?

Answer 19

Same features as insertion sequences but carry additional genes

Question 20

Transposon mechanism

Answer 20

1. Transposon binds to ends
2. Transposon is cut out
3. Chromosome is repaired but may be changes to original sequence
4. New target sequence found elsewhere and transposon inserts