homologous recombination (hr)
TRANSCRIPT
Homologous Recombination (HR)
RAGHAVENDRA N.R
PhD Scholar
Plant Breeding & Genetics
Punjab Agricultural University
Ludhiana-141004
Seminar Outline
1. Introduction
2. Mechanism of Homologous Recombination
3. Molecular basis of Homologous Recombination
4. Biological roles of Homologous Recombination
5. Practical applications
6. Conclusion
Background Knowledge
1866: Gregor Mendel – Principles of Inheritance
1900 : Bateson and Punnett –some genes can be inherited together
1913 : Morgan & Sturtevant –crossover can occur between linked genes
1931 McClintock and Creighton –crossover happens both in meiosis and mitosis
1947 : Lederberg found recombination in bacteria
1964 : Robin Holliday –Holliday Junction
1983 : Jack Szostak –DSBR pathway
Recombination can occur between similar molecules of DNA, as
in homologous recombination, or dissimilar molecules, as in non-
homologous end joining (NHEJ) or IR.
Specialized recombination called Site-specific recombination, first
characterized in prokaryotes SSR is responsible for integration of phage
genomes into the bacterial chromosomes.
Transposition allows one DNA sequence to be inserted into another
without relying on sequence homology.
Types of Recombination
Cont..
CACATGATACGTCCGATCACATTTGTTGTTCATATGTGTACTATGCAGGCTAGTGTAAA C
C
CACATGATACGTCCGATCACATTTGTTGTTCATAT
GTGTACTATGCAGGCTAGTGTAAA
A AGTATA
AA
GTATA
CACATGATACGTCCGATCACATTTGTTGTTCATATGTGTACTATGCAGGCTAGTGTAAACAACAAGTATA
GTGTACTATGCAGGCTAGTGTAAACAACAAGTATACACATGATACGTCCGATCACATTTGTTGTTCATAT
- Sequences must be the same or very similar
What is Homologous Recombination..?
Adapted from B. Alberts, et al., Molecular Biology of the Cell, Fourth edition. Garland
Science, 2002.
DNA Breaks & It’s Consequences
Internal and external, biotic and abiotic sources
Genome Stability & Environment
Waterworth et al (2011)
DNA damage response and chromatin remodelling activity in plants
Roy S (2014)
Initiation of HR at the Molecular Level
Bernard de Massy Annu. Rev. Genet. 2013. 47:563–99
Spo11: The Catalytic Activity for
Double-Strand Break Formation
DSB formation requires additional
proteins whose activities are not yet
characterized.
Recombinase as RecA/
Rad51/ Dmc1 bind to the ss-
DNA
Accessory factors as Rad54,
Rad54B, and Rdh54 help
recognize and invade the
homologous region
Cont..
Bernard de Massy Annu. Rev. Genet. 2013. 47:563–99
After the formation of
D-loop, DNA
polymerase involved to
elongate the 3’ invading
single strand
Cont..
Bernard de Massy Annu. Rev. Genet. 2013. 47:563–99
Key steps of HR
1. Alignment of two homologous DNA molecules
2. Introduction of breaks in the DNA
3. Formation of initial short regions of base pairing
between the two recombining DNA molecules.
4. Strand invasion.
5. Formation of Holliday junction & Cleavage of the
Holliday junction
Two Models For Homologous
Recombination
1.The Holliday Model
2.The Double-Strand Break Repair Model(More accurately describes recombination processes)
The Holliday Model
Homologous duplex
Single strand breaks
Cont..
Strand invasion
Branch migration
Cont.. Holiday Junction
Holiday Junction cleavage
Cont..
Resolution
patch
recombinantsplice
recombinant
The Double-Strand Break Repair Model
Cont..
Prokaryotic Recombination
Nature 374, 375
(1995)
A simple model of a
RuvA/RuvB/DNA
complex_electron
microscopy
Programmed generation of double-stranded
DNA breaks occurs during meiosis
Mechanism of cleavage by Spo11
Homologous Recombination in
Eukaryotes
DNA dynamics during meiosis
Homologous Recombination
in Eukaryotes
Recombination
Resolution
PROKARYOTIC AND EUKARYOTIC PROTEINS CATALYST REQUIRED FOR HR
Adopted from: Molecular Biology of the Gene, (2013) 7th
Ed, Benjamin Cummings, London.
Pairing homologous DNAs and strand
invasion
E. coli Protein Catalyst Eukaryotic Protein
Catalyst
Recombination Step RecA protein Rad51
Dcm1 (in meiosis)
Introduction of DSB None Spo11 (in meiosis)
HO ( for mating-type
switching)
Processing DNA breaks to generate
single strands
for invasion
RecBCD
helicase/nuclease
MRX protein (also
called Rad50/58/
60 nuclease)
Assembly of strand-exchange
proteins
RecBCD and RecA Rad52 and Rad59
Holliday junction recognition and
branch migration
RuvAB complex Not well characterized
Resolution of Holliday junctions RuvC Rad51c–XRCC3
complex, WRN, and BL
Recombination repair DNA breaks
Homologous recombination
ResectionRad50, Mre11,
Xrs2 complex
Strand invasion
Rad52
Rad51; BRCA2
DNA synthesis
Ligation, branch migration,
Holliday junction resolution
DSB
Non-homologous end-joining
Ku70, Ku80
Ligation
DSB
DNA-PKcs
Rad50, Mre11,
Xrs2 complex “Cleaning up”
of ends
XRCC4/
Ligase IV
1. Gene knockouts/Point mutations & Generating new gene/allele
combinations fueling incredible Genetic diversity.
2. Ensures proper segregation of homologous chromosome pairs
(Meo-I)
3. Preservation of Replication forks, restart replication (Escort)
telomere maintenance.
4. Ensures genomic stability of the organism.
5. Repairs damaged DNA rendered due to External & Internal
factors.
6. Gene Conversion Accounts for Interallelic Recombination
7. Genetic disorders.
Biological roles of Homologous
Recombination
INHERITED HUMAN DISEASES CAUSED BY DEFECT IN
DNA REPAIR
BREAST CANCER (BRCA1)
BERLIN BREAK SYNDROME (BLM)
ATAXIA (ATM)
XERODERMA PIGMENTATION (XPA)
FANCONI ANAEMIA (FA)
BLM FA
AT XP
This was the most unkindest cut of all…
Then, I and you and all of us fell down,
Whilst bloody flourished over us
Practical Applications
1. Used to map genes on chromosomes
2. Making transgenic cells and organisms.
3. Homology-dependent gene silencing/ Reverse breeding /Alien
Introgression / Heterosis.
4. Exploring gene sequence and its function.(Functional Genomics)
5. Gene Targeting in mammals & plants.
6. Engineering by HR: Recombineering! [CRISPRs/Cas, ZFN,
TALENs]
An endogenous gene can be replaced by a transfected
gene using homologous recombination.
Or Any Gene Modification via.. HR.
Gene Targeting
GENERAL STRATEGY FOR GENE TARGETING IN MICE
Cont..
Gene Targeting
A Novel Model System for Plant Development
and Genomic Studies
Physcomitrella patens
Schaefer (1997) Efficient gene targeting in the
moss Physcomitrella patens
Kempinet et al (1997) Targeted disruption
in Arabidopsis.
Targeted genome engineering has emerged as an alternative to
classical plant breeding and transgenic (GMO) methods to
improve crop plants and ensure sustainable food production.
The ability to precisely alter the DNA information in living cells
at loci/sites of our own choice
Genome Editing!!!
The ability to introduce
targeted, tailored changes
into the genomes of
several species will make
it feasible to ask more
precise biological
questions.
Conclusion
Hunger kills more than Ebola, but it’s not
considered a significant problem
Since, rich people can’t die of it.