Ch. 25 Pt. 3: DNA Recombination

What are the three distinct types of recombination

1. homologous (genetic)

2. nonhomologous end joining

3. Site-specific

homologous recombination

genetic exchange between two sequences that are nearly identical or are identical

NHEJ

nonhomologous end joining: genetic exchange between 2 DNA sequences (broken ends) with no sequence similarity or identity

Site-specific recombination:

genetic exchange that occurs at specific sequences. Those sequences vary depending on what type of exchange is happening

What are some of the functions of DNA recombination?

1. DNA repair

2. chromosome separation because without recombination the chromosomes do not separate properly

3. implement programmed genetic rearrangements

4. regulate gene expression

What are the three main phases of homologous recombination?

1. dsDNA break in one strand is converted to a dsDNA gap by exonucleases

2. Strand invasion

3. Branch migration

4. strand displacement

What happens during strand invasion in homologous recombination?

an exposed 3’ end pairs with its complement in the intact homolog and the other strand of the duplex (of the intact DNA) is displaced

What happens during branch migration during homologous recombination?

the invading 3’ OH end is extended by DNA Pol, and eventually after a second end-capture event, a DNA molecule with two crossovers is generated (Holliday intermediates). further DNA replication replaces the DNA missing from the site of the original dsDNA break

How are Holliday junctions resolved?

Holliday intermediate resolvases (RuvC) cleave the intermediate generating recombination products.

What enzyme is responsible for homologous recombination in e. coli?

RecBCD

What are the functions of RecBCD during homologous recombination?

5’ to 3’ helicase and nuclease

What boat-component like structure of RecC structurally splits the strands of DNA?

The rudder

How does RecBCD function in homologous recombination (through loading of RecA)?

1. dsDNa break

2. RecBCD associates

3. uses ATP to unwind and degrade the 5’ and 3’ ends of the DNA

4. It unwinds until RecC binds the Chi sequence, stopping degradation of the 3’ end (5’ end degradation continues)

5. RecBCD facilitates loading of RecA recombinase

function of RecA in homologous recombination?

RecA forms a filaments around the 3’ ends the strands; it is also involved in threading the invading strand into the the complete strand during strand invasion

What is the function of RuvC during homologous recombination?

cleaves the Holliday intermediate at opposing DNA sites

does nonhomologous end joining require related sequences?

no.

what does nonhomologous end joining start with?

a dsDNA break

When is NHEJ used during the cell cycle?

when dsDNA breaks are present during a non-replicating stage of the cell cycle, and when a sister chromatid is absent

Process of NHEJ

1. dsDNA break

2. Ku protein binds both ends of the break

3. resection of the gap and other proteins associate

4. annealing of the ends

5. DNA ligase fills in the gaps

Where does site-specific recombination occur?

short stretches of specific DNA sequences

recombinases

enzymes that bind and cleave DNA at specific sites

recombination sites

specific sequences that different recombinases recognize

In research, what is the cre recombinase system used for?

a genetic tool to knock-put genes in mice

What does Cre recombinase do?

binds the Holliday intermediate at LoxP sites and promote recombination there

What AA do recombinases have in their active sites?

tyrosine

What is the significance of having Tyr in the active site of recombinases?

Acts as a nucleophile on the DNA strand and acts as a linkage between the protein and the DNA

Basic steps of rebominase mediated recombination

1. two separate dsDNA strands in the enzyme

2. enzyme cleaves one strand from each piece of DNA

3. The single strands rejoin with the single strand from the other DNA original piece

4. Isomerization

5. Cleavage again (of the other strands from the original DNA)

6. rejoining again

Types of genomic rearrangement that can result from site specific recombination?

• inversion

• deletion and insertion

simple transposons

possess only the sequences needed for transposition including the coding sequence for the transposase

complex transposons

include everything needed for transposition and ALSO SOMETHING ELSE

who is the mother of transposable elements?

Barbara McClintock

What genetic structure does a transposon have at its ends?

terminal repeats

During transposition, what does the transposase enzyme initially do the the target DNA?

makes staggered cuts in the target site

Where is the transposon inserted?

into the site of the cuts that the transposase just made in the target DNA

What happens after the transposon is inserted into the target site of the DNA?

replication fills in the gaps, duplicating the sequences flanking the transposon

Direct transposition?

The transposon exits the original chromosome and inserts itself into the new chromosome → the original chromosome seals back up and now lacks the transposon

Replicative transposition?

the entire transposon is replicated into ta new chromosome, leaving a copy behind in the original chromosome

what is antibody diversity generated by?

unique combinations of V-J-C DNA segments using site specific recombination