Mammalians homologs of RecA proteins are called Rad51– why is this important? Box 11-1-1 Mutations in BRCA1/2 and Rad51 are associated with breast cancer BRCA1/2 proteins are part of the DNA repair proteins that are recruited to double-stranded breaks. 1

Rad52 proteins Rad52 Functions: Box 11-1-1 Rad52 proteins Rad52 Functions: -Displaces single-stranded binding proteins allowing the binding of Rad51 -Promotes the annealing of complementary single-strands 2

Localization of Repair Proteins Repair proteins are usually made at high levels and dispersed throughout the nucleus Cell partially treated by X-rays A – DAPI B – BudR incorporation C – Mre11 Antibodies

HR Resolution Box 11-1-1 Box 11-1-1 4

Holiday Junctions RuvA RuvB Box 11-1-1 Holiday Junctions RuvA Box 11-1-1 RuvB Cleavage Vertically or Horizontally by an endonuclease RuvC in E. coli, resolvase in eukaryotes 5

HR Resolution Box 11-1-1 Holiday Junctions Box 11-1-1 6

HR in Meiosis Spo11 cleaves both strands, similar to a topoisomerase Mre11, specialized nuclease chews back the 5’ end leaving a 3’ overhang

HR in Meiosis 90% in humans 10%

HR in Meiosis Meiosis yields equal number of gametes 2 Blue and 2 Red Meiosis doesn’t always yields equal numbers of gametes 3 Blue and 1 Red

HR in Meiosis Heteroduplex – a region of DNA helix formed from 1 paternal and 1 maternal strand Are they identical?

Mismatch Repair in HR Gene Conversion caused by Mismatch Repair Can Result in Loss of Heterozygosity

Mismatch Repair in HR Mismatch Repair prevents recombination between poorly matched DNA sequences

HR Movie https://www.youtube.com/watch?v=86JCMM5kb2A

Double-stranded breaks Double-stranded break are disastrous for the cell dsDNA breaks are repaired by Homologous recombination Nonhomologous end joining (NHEJ)

Nonhomologous end joining (NHEJ) NHEJ is messy and error prone NHEJ is also very complicated! Ku 70/80 are proteins that bind to the ends of the ds break Overhangs are created Ligase can seal the strands In the processing step, sequences can often deleted. Meiosis yields equal number of gametes 2 Blue and 2 Red VDJ recombination C is lost! GTTGG is lost! NHEJ repairs double-stranded breaks but results in mutations.

NHEJ Movie https://www.youtube.com/watch?v=31stiofJjYw

Nonhomologous end joining (NHEJ) is used to make mutations in genes! Why would researchers want to make mutations in genes? To study loss-of-function phenotypes. How do scientists make mutations? VDJ recombination Add mutagens like the 5-Bromouracil Ionization radiation CAS9/CRISPR Transposons

Genome Editing

Next Generation of Genome Editing? CRISPR-Cas9 -Cas9 protein -guide RNA

CRISPR/CAS9 is a technique that induced double-stranded breaks at specific sequences How is the double stranded break repaired?

CAS9/CRISPR: Genome Editing Generate Mutations in specific genes using NHEJ repair Gene Therapy: Replace “Bad” genes with “Good” genes using HR repair

CAS9/CRISPR: Genome Editing Target RNA Cas9 Protein * TARGET DNA sequence

CAS9/CRISPR: Genome Editing To get the CAS9/CRISPR system to work, you need to make cells produce the Cas9 enzyme and the target/guide small RNA. What type of promoter would you use to drive the expression of Cas9 enzyme? What type of promoter would you use to drive the expression of the guide RNA? Target RNA Cas9 Protein * TARGET DNA sequence

Types of RNA polymerases transcribe different types of genes Poly II CAS 9 protein is under a Poly II promoter Target/Guide RNA protein is under a Poly III promoter. * Finding Poly III core promoter sequences was a major challenge in this research! Poly III Target RNA Cas9 Protein https://www.youtube.com/watch?v=ZImVkl8QTW8 * TARGET DNA sequence

CRISPR/Cas9 Technologies uses both NHEJ and HR If the double-stranded DNA break is repaired by NHEJ, mutations are induced in the gene you are interested in studying!

CRISPR/Cas9 Technologies uses both NHEJ and HR If the double-stranded DNA break is repaired by NHEJ, mutations are induced in the gene you are interested in studying! If you add a “repair” template with homologous sequences, then homologous recombination is used to repair the double-stranded break. This is how gene editing occurs: “replacing the good gene with the bad gene”

Homologous Recombination