Recombination December 6, 2017

Homologous Recombination Breaks arising during DNA Replication Accurately Repairing double-stranded Breaks Exchange of Genetic Information between 2 chromosomes It only takes places between DNA duplexes that have extensive regions of sequence similarity (homology)

HR Requires Sequence Homology

HR in DNA Replication Nick in a single strand Replication Fork reaches the nick Strands separate Exonuclease degrades 5’ end

HR in DNA Replication Strand Invasion Strand Breakage DNA synthesis Replication fork Re-established

HR in DNA Replication

Ionizing Radiation Ionizing radiation induced double-stranded breaks X-rays From Slide-Player Ionizing –remove electrons

Double-stranded Breaks dsDNA breaks are caused: Directly, by ionizing radiation or other DNA damaging reagents If unrepaired, they can result in Chromosomal Abnormalities

Homologous Recombination Homologous recombination is one mechanism used to repair double-stranded DNA breaks DNA repair proteins are recruited to the double-stranded break 3’ overhangs are created Rad51 bings to the single stranded overhangs Promotes “strand-invasion” on a the “homologous” chromosome. The strand-invasion creates new sites for DNA synthesis After DNA synthesis there are junctions that must be repaired.

Figure 11-4a 12

Generation of 3’ Overhangs Figure 11-4b 13

Strand Invasion Figure 11-4c 14

New Regions of DNA synthesis Figure 11-4d 15

Resolution Figure 4-11e 16

Homologous Recombination RecA/RAD51 promotes “strand invasion”

Homologous Recombination ssDNA is generated from the double-stranded break RecA/RAD51 promotes “strand invasion” Strand invasion creates new primer::template junctions that can be synthesized with DNA polymerase

RecA/Rad51 proteins RecA/Rad51 proteins form long filaments Box 11-1-1 RecA/Rad51 proteins form long filaments Higher-order nuclear structures in Rad51-overexpressing cells. (A) Immunofluorescence staining of growth-arrested TGR928.1-9 cells reveals discrete nuclear foci and higher-order structures of overexpressed Rad51 protein (green) in a high percentage of cells. Nuclei are counterstained with DAPI (blue). Bar, 10 μm. (B) Ultrathin cross (left-hand side) and longitudinal sections (right) of nuclear Rad51 structures in TGR928.1-9 nuclei after pre-embedding immunolabeling with anti-Rad51 antibodies and 12 nm colloidal gold. Bar, 100 nm RecA/Rad51 proteins have a role in the invasion process Raderschall E et al. J Cell Sci 2002;115:153-164 19

How were Rad proteins discovered? You guessed it! E. coli Box 11-1-1 RecA protein was first identified in E. coli DNA is integrated in host chromosomes by homologous recombination is required 20

RecA/Rad51 proteins RecA protein was first identified in E. coli Box 11-1-1 RecA protein was first identified in E. coli Question: Could mutations in genes that are required for recombination be identified. Scientists made a Hfr cell that would donate Ade+ genes. Donor (Hfr): Ade+; Leu - Requires leucine supplementation Recipient (F-): Leu+; Ade- Requires adenine supplementation 21

If a recombination event occurs bacteria can grow without any RecA/Rad51 proteins Box 11-1-1 RecA protein was first identified in E. coli Question: Could mutations in genes that are required for recombination be identified. Scientists made a Hfr cell that would donate Ade+ genes. Donor (Hfr): Ade+; Leu - Requires leucine supplementation Recipient (F-): Leu+; Ade- Requires adenine supplementation If a recombination event occurs bacteria can grow without any supplementation If recombination occurred, what would be the phenotype of the recipient cell. 22

RecA/Rad51 proteins RecA protein was first identified in E. coli Box 11-1-1 RecA protein was first identified in E. coli Question: Could mutations in genes that are required for recombination be identified. Scientists made a Hfr cell that would donate Ade+ genes. Donor (Hfr): Ade+; Leu - Requires leucine supplementation Recipient (F-): Leu+; Ade- Requires adenine supplementation Randomly mutagenized the recipient cell and asked if recombination was inhibited. 23

RecA/Rad51 proteins discovery Box 11-1-1 Screened by then 2,000 mutagenize strains! Recombination event: Leu+, Ade+ Plated both the Donor (Hfr) and Recipient Strains on plates with supplementation Replica plated on plates without supplementation 24

RecA/Rad51 proteins discovery Box 11-1-1 Expected: Recombination will allow the bacteria to grow on the plates without supplementation Mutants: If bacteria have a mutation that prevents recombination, then they will not be able to grow on the plates without supplementation Which bacteria clones might have this type of mutation? Screened by then 2,000 mutagenize strains! Recombination event: Leu+, Ade+ Plated both the Donor (Hfr) and Recipient Strains on plates with supplementation Replica plated on plates without supplementation 25

RecA/Rad51 proteins discovery Box 11-1-1 Expected: Recombination will allow the bacteria to grow on the plates without supplement Mutants: If bacteria have a mutation that prevents recombination, then they will not be able to grow on the plates without supplementation. Which bacteria clones might have this type of mutation? Screened by then 2,000 mutagenize strains! Recombination event: Leu+, Ade+ Plated both the Donor (Hfr) and Recipient Strains on plates with supplementation Replica plated on plates without supplementation 26

RecA/Rad51 proteins discovery Box 11-1-1 Screened by then 2,000 mutagenize strains! Recombination event: Leu+, Ade+ Plated both the Hfr and Recipient Strains on plates with supplemented Replica plated on media without supplementation 27

Box 11-1-1 RecA/Rad51 proteins RecA monomer has more than one DNA-binding site so it can bind either ssDNA or dsDNA -It helps to catalyze strand invasion and hyrbridization 28