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.

References
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18:99-113. 2008.
2. Hong, Soogil et al. The Logic and Mechanism of Homologous Recombination Partner Choice. Molecular Cell.
51:440-453. 2013.
3. Van Gent, Dik et al. Chromosomal stability and the DNA double-stranded break connection. Nature Reviews
Genetics. 2:196-206. 2001.
4. Horodyski, Frank. MCB 7200 Lecture. Ohio University, Fall 2013.
5. Alberts, Bruce. “Chapter 6: Homologous Recombination” Essential Cell Biology, 3rd edition.
6. Filippo, Joseph, Patrick Sung, and Hannah Klein. Mechanism of Eukaryotic Homologous Recombination.
Annual Reviews in Biochemistry. 77:229-257. 2008.
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March 21, 2012. www.sciences360.com
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Gene Transfer in Bacterial Speciation. Methods of Molecular Biology. 532:29-53. 2009.
12. Krzywinska, Elzbieta, Jaroslaw Krzywinski and Jeffrey S. Schorey. Naturally occuring horizontal gene transfer
and homologous recombination in Mycobacterium. Microbiology. 150:1707-1712. 2004.
References
13. Thomas, Christopher M. and Kaare M. Nielsen. Mechanisms of, and Barriers to, Horizontal Gene Transfer
between Bacteria. Nature Reviews Microbiology. 3:711-721. 2005.
14. Dillingham, Mark S. and Stephen C. Kowalczykowski. RecBCD Enzyme and the Repair of Double-Stranded
DNA Breaks. Microbiology and Molecular Biology Reviews. 72:642-671. 2008.
15. Claverys, JP, B. Martin and P. Polard. The genetic transformation machinery: composition, localization, and
mechanism. FEMS Micriobiology Reviews. 33:643-656. 2009.
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telomere integration. Microbes and Infection. 13:731-741. 2011.
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432:187-193. 2004.
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biologist’s view. Microbiological Molecular Biology Reviews. 76:217-228. 2012.
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choice. Cell Research. 18:134-147. 2008.
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functions. Nature Reviews Molecular and Cellular Biology. 7:739-750. 2006.
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