Transcript DNA double-strand break repair

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 “nonMendelian” 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)
•
•
From Cold Spring Harbor Laboratory Press, © 2007.
5’-GCTGGTGG-3’
RecA protein is loaded by RecBCD
onto the nicked strand 5’ of the Chi
site and the complex disassembles (14)
The RecBCD Pathway
From Cold Spring Harbor Laboratory Press, © 2007.
•
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.
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
1.
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12.
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18:99-113. 2008.
Hong, Soogil et al. The Logic and Mechanism of Homologous Recombination Partner Choice. Molecular Cell.
51:440-453. 2013.
Van Gent, Dik et al. Chromosomal stability and the DNA double-stranded break connection. Nature Reviews
Genetics. 2:196-206. 2001.
Horodyski, Frank. MCB 7200 Lecture. Ohio University, Fall 2013.
Alberts, Bruce. “Chapter 6: Homologous Recombination” Essential Cell Biology, 3rd edition.
Filippo, Joseph, Patrick Sung, and Hannah Klein. Mechanism of Eukaryotic Homologous Recombination.
Annual Reviews in Biochemistry. 77:229-257. 2008.
Haber, James E., Gregorz Ira, Anna Malkova, and Neal Sugawara. Repairing a double-stranded chromosome
break by homologus recombination: revisiting Robin Holliday’s model. Phil. Trans. R. Soc. Lond. 359:79-86.
2004.
Thomas-Zucker, Julie. “Chromosomal Crossover how Genetic Exchange Increases Variation” Sciences 360.
March 21, 2012. www.sciences360.com
Francone, Frank D., Markus Conrads, Wolfgang Banzhaf, and Peter Nordin. Homologous Crossover in
Genetic Programming.
Andersson, J.O. Lateral Gene Transfer in Eukaryotes. Cellular and Molecular Life Sciences. 62:1182-1197.
2005.
Lawrence, Jeffrey G. and Adam C. Retchless. The Interplay of Homologous Recombination and Horizontal
Gene Transfer in Bacterial Speciation. Methods of Molecular Biology. 532:29-53. 2009.
Krzywinska, Elzbieta, Jaroslaw Krzywinski and Jeffrey S. Schorey. Naturally occuring horizontal gene transfer
and homologous recombination in Mycobacterium. Microbiology. 150:1707-1712. 2004.
References
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23.
Thomas, Christopher M. and Kaare M. Nielsen. Mechanisms of, and Barriers to, Horizontal Gene Transfer
between Bacteria. Nature Reviews Microbiology. 3:711-721. 2005.
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.
Claverys, JP, B. Martin and P. Polard. The genetic transformation machinery: composition, localization, and
mechanism. FEMS Micriobiology Reviews. 33:643-656. 2009.
Arbuckle, Jesse H. and Peter G. Medveczky. The molecular biology of human herpesvirus-6 latency and
telomere integration. Microbes and Infection. 13:731-741. 2011.
Singleton et al. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature.
432:187-193. 2004.
Savir, Y. and T. Tlusty. RecA-mediated homology search as a nearly optimal signal detection system.
Molecular Cell. 40:388-396. 2010.
Smith, GR. How RecBCD enzyme and Chi promote DNA break repair and recombination: a molecular
biologist’s view. Microbiological Molecular Biology Reviews. 76:217-228. 2012.
West, S.C. Molecular views of recombination proteins and their control. Nature Reviews Molecular and
Cellular Biology. 4:435-445. 2003.
Shrivastav, M., L.P. De Haro and J. A. Nickoloff. Regulation of DNA double-strand break repair pathway
choice. Cell Research. 18:134-147. 2008.
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