Pratik Shriwas
John Elmore
Quyen Luong
MCB 7200
December 3rd 2015
Origins
 1900s : Certain genes are genetically linked
 1911 : Crossover occurs between linked genes
 1931 : Crossover occurs during meiosis and mitosis
 1947 : Genetic recombination in bacteria
 1964 : Proposed model of Holliday junction
 1980 and beyond: Several models of Homologous
recombination in different species
Lobo and Shaw (2008) ; Matos and West (2014)
Homologous Recombination (HR)
 It is the biological process of genetic exchange
between two similar or identical nucleic acids
 Archaea, Eukarya and Bacteria as well as viruses
 Enzymes involved have homologous domains and are
evolutionarily conserved
 Mitosis, Meiosis and Horizontal gene transfer
Pérez-Losada, et. al. (2015); Matos and West (2014)
Bacteria
Horizontal Gene transfer
DNA repair
 Integration of donor DNA
 UV or other radiation and
into recipient cell
 Transformation
 Transduction
 Conjugation
chemical mutagens
 Double stranded breaks
Vox, M. (2009) ; Hanada and Yamaoka (2014)
Eukaryotes
Before Mitosis
Meiosis
 Occurs during cell division
 Occurs during prophase I
after DNA replication
 During Interphase (S and G2)
 Mitotic crossover
 Repair Double stranded
breaks (DSBs)
 Chromosomal crossover
Walsh, C.S. (2015)
 Genetic diversity with newer
combination of genes
Viruses
 DNA - DNA recombination (DNA viruses)
 RNA - RNA recombination (RNA viruses)
 Genetic diversity – Viral evolution
Pérez-Losada, et. al. (2015)
Improper HR
Failed HR
Improper segregation and
nondisjunction
Inefficient DSB repair
More or few chromosomes
Down Syndrome
Cancer and other related diseases
Strachan and Read (2011) ; Walsh, C.S. (2015)
Double Stranded Break – Initiation
BLM
Mimitou, et al, 2008
DSB Resectioning – A Closer look
Spo11
CtIP
NB
S1
Rad
50
Mre11
MRN
BLM
BLM
CtIP
MRN
Mimitou, et al, 2008;
Presynaptic Complex
- RPA
- RAD51
T
I
M
E
- RAD52
- BRCA2
- RAD54
D-Loop
‘Strand Invasion’
Mimitou, et al, 2008; Kowalczykowski, 2015; Fillipo et al, 2008; Renkawitz et al, 2014
DSB – Three Possible Fates
Mimitou, et al, 2008
=
Double Holiday Junction
BLM
GEN1
/YE
N1
MUS81
-EME1
MUS81
-EME1
Non-Crossover
GEN1
Resolution
Crossover
products
Mimitou, et al, 2008; Fillipo, et al, 2014; Kowalczykowski, 2015
HR Applications
 Transgenic knockout animals
 Chimeric proteins
 Anti-cancer therapy
Genetically Modified Organisms
Homologous Recombination (HR)
Non-HR
 Gene targeting knockout mice:
 Deliver artificial genetic material into mouse embryonic
stem cells
 Replace the targeted genes with homologous
recombination
 Breeding steps knockout mice
Chimeric Proteins
 Synthetic protein of two
proteins with >70% similarity
 Structure and function are
preserved
 Point mutagenesis alters
function with increasing
amino acid substitution
 Study of protein structure and
function
Carbone et al., 2007
Cancer Therapy
 Non-homologous end joining
(NHEJ) and HR to repair doublestranded breaks
 NHEJ applies to all normal cells as
well
 Cells use HR to repair DNA
double-stranded breaks resulted
from anti-cancer treatment (e.g.
radiotherapy)
 HR inhibitors prevents tumor cells
from repairing DNA breaks.
Chernikova et al. 2012
Synthetic Lethality
 Inhibiting compensatory pathways in HR-deficient
tumor cells can increase cell death (increase
effectiveness of treatment).
DNA damage (Single-stranded breaks or base damage)
Double Stranded Breaks
Poly(ADP-ribose) polymerase or PARP
HR
DNA damage (Single-stranded breaks or base damage)
Double Stranded Breaks
BRCA1/2 mutations
PARP inhibitors
Poly(ADP-ribose) polymerase or PARP
HR
Key Points
 1. Biological processes in species with HR, improper
HR
 2. RAD51 binds ssDNA and causes ‘strand invasion’ 
D-loop formed  Double Holiday Junction formed
DNA polymerase fills gaps in break  dHJ resolved to
form either crossover or non-crossover products
 3. HR can be used in gene manipulation, protein
functional studies, and therapy for certain tumors.
 4. Synthetic lethality can be used effectiveness of anticancer drugs
References
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Lobo, I., & Shaw, K. (2008). Thomas Hunt Morgan, genetic recombination, and gene mapping. Nature Education, 1(1), 205.
Matos, J., & West, S. C. (2014). Holliday junction resolution: Regulation in space and time. DNA repair, 19, 176-181.
Hanada, K., & Yamaoka, Y. (2014). Genetic battle between Helicobacter pylori and humans. The mechanism underlying
homologous recombination in bacteria, which can infect human cells. Microbes and Infection, 16(10), 833-839.
Vos, M. (2009). Why do bacteria engage in homologous recombination?.Trends in microbiology, 17(6), 226-232
Walsh, C. S. (2015). Two decades beyond BRCA1/2: Homologous recombination, hereditary cancer risk and a target for
ovarian cancer therapy.Gynecologic oncology, 137(2), 343-350.
Pérez-Losada, M., Arenas, M., Galán, J. C., Palero, F., & González-Candelas, F. (2015). Recombination in viruses:
mechanisms, methods of study, and evolutionary consequences. Infection, Genetics and Evolution, 30, 296-307.
Strachan, Tom; Read, Andrew (2011). Human molecular genetics (4th ed.). New York: Garland Science. ISBN 9780815341499.
Chernikova, S.B., Game, J.C., Brown, J.N. Inhibiting Homologous Recombination for Cancer Therapy. Cancer Biology and
Therapy, 13:2, 61-69, 2012
Carbone, M.N., Arnold, F.H, Engineering by homologous recombination: exploring sequence and function within a
conserved fold. Current Opinion in Structural Biology, 17:454-459, 2007
Kowalczykowski, S.C., An Overview of the Molecular Mechanisms of Recombinational DNA Repair. Cold Spring Harbor
Perspectives in Biology, 7:a016410, 2015
http://www.bio.davidson.edu/courses/genomics/method/homolrecomb.html
Mimitou, E.P., Symington, E.P. Nucleases and Helicases take Center Stage in Homologous Recombination, Trends in
Biochemical Sciences. Vol. 34, No. 5, 2009
Renkawitz, et al. Mechanisms and principles of homology search during recombination, Nature: Molecular and Cell Biology,
15: 369, 204
Fillipo, J.S., Sung, P. Klein, H, Mechanism of Eukaryotic Homologous Recombination, Annual Review of Biochemistry,
77:229-57, 2008