Mechanisms of Homologous Recombination

Pratik ShriwasJohn Elmore

Quyen LuongMCB 7200

December 3rd 2015

Origins1900s : Certain genes are genetically linked1911 : Crossover occurs between linked

genes1931 : Crossover occurs during meiosis and

mitosis1947 : Genetic recombination in bacteria1964 : Proposed model of Holliday junction1980 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

transferDNA repair

Integration of donor DNA into recipient cell

TransformationTransductionConjugation

UV or other radiation and chemical mutagens

Double stranded breaks

Vox, M. (2009) ; Hanada and Yamaoka (2014)

Eukaryotes Before Mitosis

MeiosisOccurs during cell

division after DNA replication

During Interphase (S and G2)

Mitotic crossoverRepair Double stranded

breaks (DSBs)

Occurs during prophase I

Chromosomal crossover Genetic diversity with

newer combination of genes

Walsh, C.S. (2015)

VirusesDNA - DNA recombination (DNA viruses) RNA - RNA recombination (RNA viruses) Genetic diversity – Viral evolution

Pérez-Losada, et. al. (2015)

More or few chromosomesDown Syndrome

Improper segregation and nondisjunction

Inefficient DSB repair

Cancer and other related diseases

Failed HR

Improper HR

Strachan and Read (2011) ; Walsh, C.S. (2015)

Double Stranded Break – Initiation

BLM

Mimitou, et al, 2008

DSB Resectioning – A Closer look

Mre11

Rad50

NBS1

CtIP

CtIP

MRN

MRN

BLM

BLM

Spo11

Mimitou, et al, 2008;

Presynaptic Complex

- RAD51

- RAD52

- RPA

- BRCA2

- RAD54

D-Loop

TIME

‘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=

MUS81-

EME1

GEN

1

BLM/YEN1

GEN

1

Non-Crossover Crossover products

MUS81-

EME1

Resolution

Mimitou, et al, 2008; Fillipo, et al, 2014; Kowalczykowski, 2015

HR ApplicationsTransgenic knockout animalsChimeric proteinsAnti-cancer therapy

Genetically Modified Organisms

Gene targeting knockout mice: Deliver artificial genetic material into mouse

embryonic stem cellsReplace the targeted genes with homologous

recombinationBreeding steps knockout mice

Homologous Recombination (HR)

Non-HR

Chimeric ProteinsSynthetic 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 TherapyNon-homologous end joining

(NHEJ) and HR to repair double-stranded 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).

Poly(ADP-ribose) polymerase or PARP

DNA damage (Single-stranded breaks or base damage)

HR

Poly(ADP-ribose) polymerase or PARP

DNA damage (Single-stranded breaks or base damage)

HR

BRCA1/2 mutationsPARP inhibitors

Double Stranded Breaks

Double Stranded Breaks

Key Points1. Biological processes in species with HR,

improper HR2. 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 anti-cancer drugs

References1. Lobo, I., & Shaw, K. (2008). Thomas Hunt Morgan, genetic recombination, and gene mapping. Nature

Education, 1(1), 205.2. Matos, J., & West, S. C. (2014). Holliday junction resolution: Regulation in space and time. DNA repair, 19, 176-

181.3. 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.

4. Vos, M. (2009). Why do bacteria engage in homologous recombination?.Trends in microbiology, 17(6), 226-2325. 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), 343-350.6. 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, 296-307.7. Strachan, Tom; Read, Andrew (2011). Human molecular genetics (4th ed.). New York: Garland

Science. ISBN 9780815341499. 8. Chernikova, S.B., Game, J.C., Brown, J.N. Inhibiting Homologous Recombination for Cancer Therapy. Cancer

Biology and Therapy, 13:2, 61-69, 20129. 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, 200710. Kowalczykowski, S.C., An Overview of the Molecular Mechanisms of Recombinational DNA Repair. Cold Spring

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Biochemistry, 77:229-57, 2008