DNA recombination

Homologous Recombination Conjugation Transformation Transduction Site-specific Recombination Transposition

1.1 homologous Recombination

Homologous recombination occurs between identical or nearly identical sequences. It is also called general recombination.

5 3 3 5

3 5 endonuclease 5 (recBCD) 3

5 3 3 5

5 3

3 5 5 3

DNA invading (recA)

5 3 3 5 3 5 5 3

5 3 3 5

3 5 5 3 endonuclease (recBCD) 3 5 5 3

Branch migration (recA)

5 3 3 5

DNA ligase

5 3 3 5

3 5 5 3

Holliday intermediate

Holliday intermediate
5 3 3 5 3 5 5 3

3 5

3 5

endonuclease (ruvC)
3 5 5 3

5 3 3 5

3 5

endonuclease (ruvC)
3 5

5 3 3 5

5 3

5 3

DNA ligase
5 3 3 5

5 3

3 5 3 5

DNA ligase patch recombinant

5 3 5 3

splice recombinant
3 5

5 3

3 5

1.2 Conjugation
Bacterial Conjugation -transmission of genetic information from a donor bacterium to a recipient cell through cell-to-cell contact.

Conjugation process

Conjugation process

Conjugation process

1.3 Transformation

Introduction of an exogenous DNA into a cell, causing the cell to acquire a new phenotype.

Transformation
Genetic recombination in which a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and it is exchanged for a piece of the recipient's DNA. Involves 4 steps

The 4 steps in Transformation

1. A donor bacterium dies and is degraded

2. A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium

3. The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA

4. Exchange is complete

Transformation

DNA

Transformation experiment of Streptococcus pneumoniae

1.4 Transduction
-transfer of DNA fragments from one bacterium to another bacterium by a bacteriophage.

Structure of T4 bacteriophage

Contraction of the tail sheath of T4

What are Bacteriophages?

Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of the host biosynthetic machinery.

Transduction

Two types of Transduction

Generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle. Specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle

Seven steps in Generalised Transduction 1. A lytic bacteriophage adsorbs to a susceptible bacterium. 2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes 3. Occasionally, a bacteriophage head or capsid assembles around a fragment of donor bacterium's nucleoid or around a plasmid instead of a phage genome by mistake.

Seven steps in Generalised Transduction (contd)

4. The bacteriophages are released.

5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium

Seven steps in Generalised Transduction (contd)

6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium .

7. The donor bacterium's DNA is exchanged for some of the recipient's DNA.

Six steps in Specialised Transduction

1. A temperate bacteriophage adsorbs to a susceptible bacterium and injects its genome . 2. The bacteriophage inserts its genome into the bacterium's nucleoid to become a prophage.

Six steps in Specialised Transduction (contd)

3. Occasionally during spontaneous induction, a small piece of the donor bacterium's DNA is picked up as part of the phage's genome in place of some of the phage DNA which remains in the bacterium's nucleoid. 4. As the bacteriophage replicates, the segment of bacterial DNA replicates as part of the phage's genome. Every phage now carries that segment of bacterial DNA.

1.5 Site-specific Recombination

Site-specific recombination occurs at a specific DNA sequence. The first example was found in the integration between DNA and E. coli DNA.

DNA integration

Phase variation of Salmonella typhimurium flagella

hix
hin

hix
P P2
H2

rH1

P1

H1

DNA
H2 flagellin Hin repressor
P2
P2

hin

H2

rH1

P1

H1

H segment

H1 flagellin

Recombination signal sequence (RSS)

CACAGTG (12/23) ACAAAAACC GTGTCAC
RSS

TGTTTTTGG

Recombination activating gene enzyme (RAG1 and RAG2)

1.6 Transposition
the movement of specific pieces of DNA in the genome. resembles site-specific recombination being catalyzed by special enzymes.

IS Transposition

insertion sequences (IS) including: inverted repeats (IR) 9~41bp transposase gene repeated sequences 4~12bp

Transposase gene

types of IS transposition
duplicative transposition Conservative transposition

duplicative transposition

Conservative transposition

transposon
Insertion sequence + another gene (usually antibiotic gene)

Transposase gene

tet-R gene

Transposons Transposition