Lecture 52: Recombination and Transposons Flashcards
Wednesday 26th February 2025
Is translocation a method of horizontal gene transfer?
No
Why isn’t translocation a method of horizontal gene transfer?
Translocation is not a method of horizontal gene transfer because it involves movement within a genome rather than transfer between organisms.
What is recombination?
Breaks and joins DNA into a new combination
Allows rapid evolution quicker than accumulating mutations
Permits auxotroph to prototroph switch by complementation of deleted function
Necessary for all the methods of horizontal gene transfer, except plasmids
Why do we recombine DNA?
Creates genetic diversity
“Easy” to do in eukaryotic cells which are diploid
In bacteria requires fragments of DNA from : transformation; transduction; conjugation (plasmids or transposons)
What are the 2 types of recombination?
Homologous and non-homolgous
What is homologous recombination?
Switching DNA that is similar
e.g. same gene different allele
What is non-homolgous recombination?
Repair of double stranded DNA break by simply joining with another piece of DNA
Describe homolgous recombination
Requires extensive homology (similarity of sequence)
Can result in replacement of faulty gene
Examples of this in lecture on conjugation
Involved Holliday junctions: Model proposed in 1964 by Robin Holliday
Desvribe mechaninsm 1 of homolgous recombination
- Align 2 homolgous strands of DNA
- One strand is nicked at a specific sequence by enzyme RecBCD at a specific sequence
- The unwound 3’ end will invade the other DNA duplex
- The DNA will be stabilised by SSB protein (binding protein) and this will be catalused by enzyme RecA.
Describe RecA
- Has functional homology in ALL known organisms incl. eukaryotes and archaea
- Essential for DNA repair
- Multiple functions: binds single stranded DNA; has two binding sites so can hold two pieces of DNA together; catalyses branch migration
Describe enzyme RecBCD
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
Describe mechanism 2 of homolgous ecombination
- A nick will now also be made in the strand of Dna that was invaded.
- Both of the nicked strands of DNA will cross over, resultingi n branch migration
- Now extensive heteroduplexes form and this will be catalysed by RuvAB helicase
- Isomerisation (corssing and uncrossing of the strands) will result in the formation of a holiday junction
- Then the 2 DNA sequences will be cleaved from each other by RuvC and the holiday junction will be resolved and 2 separate DNA strands will be formed
- Repair mechanisms will resolve mismatched base pairs
How longmay the heteroduplexes be?
1000s of bp long
What can homolgous recombination rescue?
Homologous recombination can rescue a double stranded break
How does homolgous recombination rescue a double stranded break?
- Double-stranded break – A break occurs in the DNA
- 5’ to 3’ exonuclease activity – One of the DNA strands is degraded at the 5’ ends, exposing 3’ overhangs.
- Strand invasion – The single-stranded 3’ overhang invades a homologous DNA sequence (likely shown in red).
- DNA synthesis – The missing DNA is synthesized using the homologous strand as a template.
- Ligation: The newly synthesized strands are connected, forming a double Holliday junction.
- Branch migration: The junctions move along the DNA, extending the region of exchange.
- Resolution of Holliday junctions: The junctions are cleaved, completing the repair process.
This process ensures accurate repair of DNA damage by using a homologous sequence as a template, preventing mutations or chromosomal rearrangements.
Can Hfr strains transfer their genome?
Yes
Describe non-homolgous recombination (DNA strands aren’t similar)
No sequence homology
e.g Insertion and excursion of 𝝺 phage
Transposon events (site specific)
Double stranded break repair
Used in Crispr-Cas9 (biotechnology)
Non-homologous recombination after DS breaks
Strands may be ligated again, or recombination will occur with a new type of DNA
Describe insertion sequences
- Insertion sequences (IS) are small pieces (~1000 bp) of DNA that can hop from one position to another
- The hop is called transposition catalysed by transposase
- Transposase is encoded by the insertion sequence
- Carries no other genes
- Also has tandem repeat at the ends, which are needed for insertion
Describe insertion sequences and phenotypes
- Carries no novel features
- Don’t carry genes, except for those that contro; the ability to loop
- Can disrupt genes due to insertion
- High degree of reversion as IS simply moves somewhere else
- If a housekeeping gene is disrupted this will be lethal
Describe transposons
- Has same features as insertion sequences, but carry additional genes i.e resistance genes, tra genes (which are necessary for cojugation)
- Like insertion sequences they can knock-out genes through insertion
Describe the mechanism of transposon movement
- A transposase enzyme (represented as gray ovals) recognizes and binds to the ends of the transposon (green segment).
- The transposon is excised from the original DNA sequence.
- The chromosome is repaired, but this repair may introduce mutations or changes in the original sequence.
- The transposase searches for a new target site in the genome where the transposon can be inserted.
- The transposon is inserted into the new location (blue and red markings indicate the new integration site).
What is a transposon?
A jumping gene
What are the 2 types of trransposition?
Conservative (main method) and replicative
What is replicative transposition?
The original copy is retained AND a new copy is made which inserts elsewhere
Key messages
Both DNA repair and horizontal gene transfer depends on recombination
Recombination can be homologous and non-homologous
Transposons are jumping genes which can convey a phenotype but also knock out genes through insertion
