Lecture 20: Homologous Recombination and Homologous End Joining

In recombinational repair, what does replication through a single strand break cause?

the replication fork to collapse

What can double stranded breaks be caused by?

• oxidative damage

• radiation damage

• cellular nucleases

Homologous recombination is…

• important for DNA repair

• required for meiosis in eukaryotes

• present in all organisms

Why is homologous recombination important in meiosis?

crossovers between homologous chromosomes are necessary for segregation and diversity in gamates

What is the process for the double strand break model for recombination initiation?

1. Generate gapped DNA with 3’ tails

2. 3’ strand invasion and Pol I mediated DNA synthesis

3. Branch migration and generation of two holiday junctions

4. Resolution of a holiday junction through either a crossover or a patch

What are the enzymes in the primary pathway for homologous recombination?

• RecA

• RecBCD

• RuvAB

• Single strand binding protein

• Pol I

• Ligase

What are the functions of RecBCD?

• generate a 3’ overhang to allow strand invasion

• look for double stranded nicks

• contain helicase/nuclease activity

What happens when RecBCD encounters a chi site?

RecBCD stops degrading the 3’ end and degrades the 5’ end instead

True or False: Chi sites are overrepresented in the E. coli genome; these sites should be 1 in every 1/65 kb but are found every 1/4.5 kb.

true

What do chi sites help to accelerate?

the functions of RecBCD

What does RecA do?

• forms filaments on ssDNA

• finds homologous strand

• mediates strand invasion

What enzyme is required for all of homologous recombination?

RecA

What is important to know about RecA?

• recA mutants don’t do homologous recombination

• conserved

• looks through entire chromosome for homology

• works better with longer regions of homology

What is RuvAB responsible for?

branch migration of holiday junctions

What is RuvB?

an ATP-dependent helicase

What does RuvA do?

recognize and bind to the holiday junction

What does RuvC do?

cuts the holiday junction and randomly chooses the orientation

When does recombination in meiosis occur?

prior to the first mitotic division

Can homologous recombination repair a double-stranded break perfectly?

yes, but homologous recombination needs two copies of chromosomes, and only cells in the S or G2 phase have two copies

What method can repair double stranded breaks without an intact copy of the damaged DNA?

nonhomologous end joining (NHEJ)

True or False: NHEJ is the most important pathway for DSB repair in nonproliferating cells of higher eukaryotes.

true

Can NHEJ repair a double-stranded break perfectly?

no, it cannot

What does NHEJ do?

• cleans up the ends of DNA

• ligates the ends of dsDNA molecules together

True or False: There is a loss of DNA in NHEJ.

true

True or False. Most bacteria don’t have NHEJ, but those that do spend a lot of time in a stationary phase such as tuberculosis.

true

What protects the ends from NHEJ?

telomeres

If simultaneous double stranded breaks cause translocation, then what are some of the possible implications?

• Inappropriate express of Abl causes Leukemia

• Results in cell death

• Breaks in the dsDNA are swapped

What does the CRISPR/Cas 9 system do?

prevents viral infection in a bacterial cell