homologous recombination (hr)

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

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considered a significant problem

Since, rich people can’t die of it.