Mechanisms of Homologous Recombination Mark Slayton Ohio University - MCB 7200 December 3rd, 2013 Dr. Frank Horodyski

Overview Background Information What is Homologous Recombination? DNA double-strand break repair Chromosomal crossover Horizontal gene transfer The RecBCD Pathway Homologous Recombination in Eukaryotes Practical Applications

Overview Background Information What is Homologous Recombination? DNA double-strand break repair Chromosomal crossover Horizontal gene transfer The RecBCD Pathway Homologous Recombination in Eukaryotes Practical Applications

Background Information The term ‘homologous recombination’ was developed over a series of discoveries demonstrating “non- Mendelian” genetics Early 1900’s – traits can be genetically linked 1911 – “crossing-over” can occur between chromosomes 1931 – chromosomal crossover in gametes (meiosis) and in somatic cells (mitosis)

Background Information 1947 –prokaryotic genetic recombination, establishes E. coli as a model organism 1964 – meiotic recombination, Holliday junction 1983 – double-strand break repair (DSBR) pathway Additional research leads to discovery of a multitude of different pathways for homologous recombination in organisms from all three domains – bacteria, archaea, and eukarya – as well as in viruses

Overview Background Information What is Homologous Recombination? DNA double-strand break repair Chromosomal crossover Horizontal gene transfer The RecBCD Pathway Homologous Recombination in Eukaryotes Practical Applications

What is Homologous Recombination? DNA double-strand break repair Bacteria primarily employ the RecBCD pathway (“A”) Eukaryotes typically use either double-stranded break repair (DSBR) or synthesis-dependent strand annealing (SDSA) (“B”) “Repair of DNA double-strand breaks”, Dale Wigley. www.icr.ac.uk

What is Homologous Recombination? Chromosomal crossover Shuffles alleles and genetic traits to drive evolution, adaptation Incorrect rejoining of chromosomes leads to serious problems like Down’s syndrome or miscarriage in humans (8) From Classical Genetics – Chapter 9, Fundamentals of Genetics

What is Homologous Recombination? Horizontal gene transfer Horizontal/lateral gene transfer in bacteria relies on homologous recombination (13) Often used for the spread of genes conferring antibiotic resistance as well as virulence factors (12) From Furuya and Lowy, Nature Reviews Microbiology. 4, 36-45 (2006)

Overview Background Information What is Homologous Recombination? DNA double-strand break repair Chromosomal crossover Horizontal gene transfer The RecBCD Pathway Homologous Recombination in Eukaryotes Practical Applications

The RecBCD Pathway RecBCD complex binds to the blunt end of DS break The complex will pause upon recognizing the Chi site (14) 5’-GCTGGTGG-3’ RecA protein is loaded by RecBCD onto the nicked strand 5’ of the Chi site and the complex disassembles (14) From Cold Spring Harbor Laboratory Press, © 2007.

The RecBCD Pathway DNA/RecA nucleoprotein performs strand invasion on the identified homologous sequence, forming a displacement loop (D-loop) (19) Conversion to a Holliday structure leads to repair by crossing over Resolution of Holliday structure is carried out by the RuvABC complex. From Cold Spring Harbor Laboratory Press, © 2007.

Electron micrograph of unwinding DNA during RecBCD homologous repair From Smith, GR. Microbiol. Mol. Biol. Rev. 76:217-228. 2012.

Overview Background Information What is Homologous Recombination? DNA double-strand break repair Chromosomal crossover Horizontal gene transfer The RecBCD Pathway Homologous Recombination in Eukaryotes Practical Applications

Homologous Recombination in Eukaryotes Shortly after a double-strand break occurs, the MRN complex (MRX complex in yeast) initiates repair as described previously (4) Leads to formation of 3’ overhangs which are coated in Rad51, the RecA homolog (4) The invading 3’ strand is extended by DNA synthesis, forming the Holliday structure (22)

Homologous Recombination in Eukaryotes In DSBR, the non-invading DNA/Rad51 nucleoprotein is base-paired by the newly synthesized strand (22) and restored by DNA polymerase/ligase (4) Double Holliday structures produced In SDSA, only one Holliday structure is formed by the invasion of the nucleoprotein. Top strand is synthesized and ligated, resulting in a non-crossover homologous repair (23)

Overview Background Information What is Homologous Recombination? DNA double-strand break repair Chromosomal crossover Horizontal gene transfer The RecBCD Pathway Homologous Recombination in Eukaryotes Practical Applications

Practical Applications Drs. Mario Capecchi, Martin Evans, and Oliver Smithies shared the 2007 Nobel Prize in physiology or medicine for developing this technique From “Gene Modification in Mice” www.nobelprize.org 2007.

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