Ch. 14 Site Specific Recombination and Transposition Flashcards
How are site specific recombination and transposition similar? (2)
- They both recombine DNA fragments having little-to-no homology
- They are both mediated by enzymes with evolutionary relationships to topoisomerases, meaning they promote nearly isoenergetic phosphroyl transfers
What is site specific recombination?
The precise and predictable rearrangement of DNA between two specific sequences.
What kinds of changes can site specific recombination cause? (3)
Can cause inversions, deletions, or insertions.
What two things does site specific recombination involve?
It involves a short (20-200 base) sequence and a recombinase enzyme that acts at that sequence.
Which classes of cells does site specific recombination occur in?
Found in prokaryotic and eukaryotic genomes.
How does site specific recombination work/happen? (3)
- Sequences often contain inverted repeats (binding site) separated by a short core sequence (core)
- The cores of two sites align, and recombinase binds the binding sites
- The recombinase mediates recombination between the two identical cores
What is the outcome of site specific recombination determined by?
Outcome depends on the orientation of the cores. For example, cores in the opposite direction/orientation will cause an inversion.
What is in the active sites of recombinases and what occurs there (3)?
The active sites in recombinases contain tyrosine and serine residues.
1. the -OH group of the a.a. breaks phosphodiester bonds
2. after strands are broken, they are reattached to the partner strands
3. the complex rearranges and does the same for the other two strands
What are some common uses of site specific recombination in cells/organisms? (3)
- Insertion of some viral genomes into host DNA (e.g. P1 phage, ƛ phage)
- Regulation of gene expression (e.g. Salmonella’s lack of flagellin to evade immune detection)
- Used in biotechnology to invert, delete, or insert genetic sequences depending on orientation of recognition sequences (e.g. Cre-lox, Flp)
What is transposition?
The movement of a genetic sequence from one site in a genome to a different site.
How is the movement of genetic sequences by transposition described? (2)
Sometimes the movement is random. Sometimes the movement favors certain DNA sites.
Why is transposition regulated?
Transposition could disrupt an important gene and kill the cell, and therefore the transposon, so it is tightly regulated.
How are transposons thought of in biology?
Thought of as “molecular parasites” but have influences the evolution of important traits.
What are the three main pathways of transposition?
Cut and Paste, Replicative, and Retrotransposon.
How does the cut and paste method of transposition occur? (2)
- An enzyme creates a ds break that will be repaired
- the transposon is reinserted into another site of the same, or a different chromosome
Does the cut and paste method of transposition change copy number?
No. It does not increase copy number.
How does the replicative method of transposition occur? (2)
- the transposon sequence is partially cut and joined to the target sequence before excision
- DNA replication at 3’-OH ends creates two complete copies of the transposon in each site
Does the replicative method of transposition change copy number?
Yes. It will increase copy number.
How does the retrotransposon method of transposition occur? (3)
- Transposon DNA is transcribed to RNA
- transposon RNA is reverse transcribed to DNA
- DNA is inserted into a target site
What does the DNA sequence of a transposon encode for? (2)
The DNA sequence encodes the enzyme(s) that catalyze the movement. It sometimes has additional genes present.
What is the enzyme involved in transposition and what does it do (2)?
The enzyme involved in the transfer is a transposase. It performs two functions…
1. hydrolysis of phosphodiester bonds (make the cut)
2. attack of a different phosphodiester bond using the 3’-OH
How are breaks and nicks in the original strands repaired after transposition?
By DNA repair mechanisms.
What kind of transpositions usually occur in bacteria and how are they categorized?
Bacterial transposons are mostly cut and paste, and are categorized by the types of genes present.
What are the three categories of bacterial transposons? Describe each.
Insertion sequences (IS): contains only the transposase
Composite transposons: carry additional genes in addition to the transposase
Complex transposons: carry many additional genes involved in transposition AND other cellular processes (some of these are viruses)
