DNA Double-Strand Break repair by

homologous recombination
Valérie BORDE
Institut Curie, Paris

October 2nd 2023

Module MU4BM007
 Outline
- The different sources of DNA DSBs
- The possible ways to repair a DSB
- 1st step: Sensing of the DSBs
- Repair pathway choice
- Competition between 53BP1 and CtIP/Sae2 for DSB end resection
- Non-homologous end-joining (NHEJ)
- The DSB resection step
- Initial short-range resection (Sae2 + Mre11 complex)
- Long-range resection (Exo1 or Sgs1+Dna2)
- Post-resection steps and pathways
- Alternative end joining (alt-EJ)
- Single-strand annealing (SSA)
- Homologous recombination
- The different steps of homologous recombination
- Strand Invasion -> D-loop formation
- Break-Induced Replication (BIR)
- Synthesis-Dependent Strand Annealing (SDSA) -> non-crossover
- Double Holliday Junction -> dissolution or resolution

- Regulation of the pathway choice by the cell cycle stage and chromosomal context
- Homologous recombination and genome stability in cancer cells
- The special case of programmed DSBs
DNA double-strand breaks are the most dangerous DNA lesions

Mutations Chromosomal Chromosome

rearrangements loss

≈50 DSBs per cell per cell cycle occur in our cells

Ø Need of efficient ways to accurately repair DSBs

DNA double-strand breaks are the most dangerous DNA lesions

Spontaneous, Developmentally
accidental programmed

External
- Ionizing radiation
- Exposure to radiomimetic or genotoxic agents

Endogenous:
- Replication stress:
- Blockage of replication fork, accidental or due to a DNA lesion
- Byproducts of endogenous cellular metabolisms

Programmed DSBs introduced by specific DNA-cleaving enzymes:

- Programmed DNA elimination in ciliates
- RAG1-RAG2 during V(D)J recombination in developing lymphoid
cells
- Spo11 for meiotic recombination
Repair of replication failures by homologous recombination

Guirouilh-Barbat et al (2014) Frontiers Genetics

 The main pathways to repair DSBs

Spontaneous, Developmentally
accidental programmed

CtIP, MRE11
complex

NHEJ

Homologous Single-Strand Alt-EJ

Recombination Annealing
From Ceccaldi et al (2016) Trends Cell Biol
 1st event: Sensing of the double-strand break

- The Mre11 complex is the first to bind to DNA ends.

- The Ku70/Ku80 heterodimer also binds free DNA ends.

DSB

Recruitment of the Atm (Tel1) kinase

Autophosphorylation and activation of the Atm (Tel1) kinase
Phosphorylation of Mre11 complex and recruitment of other Atm targets
 2nd event: Choice how to repair the DSB

ALT-EJ

after
resection
no
resection

Symington & Gautier (2014) CSHperspectives

- How is the decision to repair by homologous recombination or NHEJ taken?

- The main event is resection
- Controlled by many factors, cell cycle stage, the cell type, the chromosomal region
DSB end resection: degrades the 5’ extremities of the DSB

DSB
5’ 3’
3’ 5’

3’
5’ 3’ Nucleolytic degradation
3’ 5’
3’ 5’ to 3’

3’
5’ 3’ 3’ overhangs
3’ 5’
3’

Ø Generates 3’ ssDNA overhangs able to invade an intact

homologous template
No resection -> The Non Homologous End Joining (NHEJ) pathway

- Modification and ligation of the broken DNA ends with no homology

DSB

NHEJ

• No need for a homologous template for repair

• Major pathway in G1 phase cells
• Can be accurate, but also often introduces 1-4 nt deletions
• Involved to repair programmed DSB in the V(D)J recombination in the immune system
The Non Homologous End Joining (NHEJ) pathway
The Non Homologous End Joining (NHEJ) pathway

XRCC4 XLF filament

Helps keep ends together

 Mechanism of DSB end resection
DSB
5’ 3’
3’ 5’

3’
5’ 3’
3’ 5’
3’

Proposed to occur in two steps:

- Short-range: The initial resection is made by the endonuclease

and exonuclease activities of the Mre11 complex, together
with Sae2 (CtIP in mammals)

- Long-range: two other pathways take over, to generate longer

3’ overhangs:
- The Sgs1 (BLM) helicase + the Dna2 flap endonuclease
- The Exo1 exonuclease
 The Mre11 complex and Sae2/CtIP

endonuclease

3’ to 5’
Nuclease domain
exonuclease

Sae2 Signalling

Fonctions not involving Mre11 nuclease activity:

- DSB signalling (activation of ATM kinase)
- Keeping DSB ends together

Symington & Gautier (2014) CSHperspectives

The Mre11 complex and Sae2/CtIP

endonuclease

3’ to 5’
Nuclease domain
exonuclease

Sae2 Signalling

Sae2

Sae2/CtIP
activates the
endonuclease
Endonuclease
activity of Mre11

Symington & Gautier (2014) CSHperspectives

Short-range resection by Mre11 complex + Sae2/CtIP

DSB
5’ 3’
3’ 5’

The Mre11 complex is recruited

5’ 3’
3’ 5’
Sae2/CtIP activates the
endonuclease activity of Mre11
3’
5’ 3’
3’ 5’
3’

The exonuclease activity of 3’

Mre11 degrades from 3’ to 5’ 5’
3’
3’
5’
3’

RPA
3’
5’ 3’
3’ 5’
3’
 Long-range resection of 5’ DSB ends

- After the initial step by Mre11 complex+Sae2/CtIP, there are two redundant
pathways:

- One by Sgs1/Top3/Rmi1 (STR) helicase and Dna2 Flap endonuclease

- The other by the Exo1 exonuclease

5’
3’ RPA
3’
3’ 5’
3’

STR Exo1
3’
5’ 3’
3’ 5’
3’
Dna2 5’

Extension of resected tracts

STR 5’ 3’
Exo1
5’ 3’
3’ 5’
3’
Dna2
and/or
 The 53BP1/Rad9 protein competes with resection factors

Ø The 53BP1 protein (Rad9 in budding yeast) counteracts resection – binds specific
histone marks close to the DSB or at telomeres
Ø Inhibits the activity of Sae2/CtIP
Ø Favors repair through NHEJ
 The Shieldin complex works with 53BP1 to
counteract resection

Ø The 53BP1 protein (Rad9 in budding yeast) binds to histones

Ø 53BP1 recruits the Shieldin complex that tends to fill the resected ends -> NHEJ

Greenberg (2018) Nature Cell Biology 20, 862-3.

 3rd step: After resection: 3 possibilities

Homologous Single-Strand Alt-EJ

Recombination Annealing

From Ceccaldi et al (2016) Trends Cell Biol

 Alternative End Joining (Alt-EJ)
- Sometimes called also “MMEJ” (Micro-Homology Mediated End Joning):
involves some processing of the ends and microhomologies at the junction

(DNA polymerase)

Ø Very mutagenic (small

deletions)

• Can occur in homologous recombination mutants, when ends have already been resected
• Also occurs in normal cells, where it competes with homologous recombination
• Repairs DSBs that occur during mitosis (2022-2023)
 Single-Strand Annealing (SSA)

- Sometimes, resection will expose single-stranded regions that are complementary

to each other (direct repeats). These will reanneal, generating a deletion between
the repeats.
This is called SSA (Single-Strand Annealing) and is highly mutagenic.

DSB
5’ 3’
3’ 5’

resection

Strand annealing between the repeats (Rad52)

Cleavage by Rad1-Rad10 (XPF-ERCC1)

Ligation by DNA Ligase I

Deletion product
 Homologous Recombination: involves strand invasion by Rad51

Loading of Rad52/BRCA2 and Rad51

Formation of the Rad51 filament

with help of the Rad51 accessory
factors

Removal of Rad52/BRCA2

Strand invasion
with the help of Rad54 D-loop structure
 The Rad51 recombinase
- Homolog of RecA in procaryotes
- There is a meiosis-specific version, called Dmc1
- Binds adenosine triphosphate (ATP) via a Walker box
- Oligomerizes on both single-strand DNA (ssDNA) and double-strand
DNA (dsDNA) forming right-handed nucleoprotein filaments
Many factors promote the function of Rad51
(Rad51 mediators)
 Rad52 (budding yeast) and BRCA2 (human and plants)
- Promote the loading of Rad51 and the replacement of RPA
- Essential for homologous recombination
- Rad52 also has strand annealing activities, both in yeast and humans
 The Rad51 paralogs

- Some sequence similarity with Rad51

- Not active for strand invasion
- Promote the growth / stability of the Rad51 filament

Ø Rad55-Rad57 (budding yeast)

Ø RAD51B-RAD51C-RAD51D/XRCC2-XRCC3 (human)
 The PCSS/Shu complex

- Not strong sequence homology to Rad51 but structural homology

- Not active for strand invasion
- Promote the growth / stability of the Rad51 filament

- Psy3-Csm3-Shu1-Shu2 (budding yeast)

- SWS1-SWSAP1 (human)

DNA
binding
 The PCSS/Shu complex

- The Psy3-Csm2 heterodimer structurally mimmics a Rad51 dimer

Sasanuma et al (2013) Nature Communications

Factors that antagonize Rad51 filament formation / stability

The Srs2 helicase

 Factors that antagonize Rad51 filament formation / stability
The FIGNL fidgetin AAA ATPase

- In Arabidopsis
- In human cells

Is able to dissociate Rad51 from ssDNA

Its mutation is able to restore Rad51 filament formation and homologous recombination to
Rad51 mediator mutants:
- BRCA2-/- mutants (in Arabidopsis thaliana)
- RAD51 paralogue SWSAP1 - deficient human cells
Ø SWSAP1 protects RAD51 filaments from the action of FIGNL1

Structural similarity
-> competition Kumar et al (2019) Nucleic Acids Res
Matsuzaki et al (2019) Nature Communications
 Factors that promote the D-loop formation

Rad54: ATP-dependent
translocase
Chromatin remodellers family

D - loop
The different homologous recombination pathways after strand invasion
D-loop

Symington et al (2014) Genetics

Break-Induced Replication occurs when one end of the DSB is lost

D-loop

• At a broken replication fork

• Close to a chromosome end

• A unidirectional replication fork is

re-established
• The DNA polymerases used are
mutagenic
• “conservative” replication
-> a whole chromosome arm can
be copied
 The SDSA (Synthesis-Dependent Strand Annealing) pathway leads to
non crossover products and is predominant in somatic cells

D-loop
Helicases:
BLM/Sgs1
R-TEL
Srs2

- The regulation of this step is not well known in somatic cells

- Action of helicases that dismantle D-loops, and Rad51

filaments (Srs2, BLM/Sgs1, R-TEL…)

- It suppresses crossovers and loss of heterozygosity

-> preserves genomic integrity
 Second end capture and formation of the double-Holliday
junction

DSBR (Double-Strand
Break Repair model) D-loop
Szostak et al (1983) Cell
Strand annealing by Rad52

DNA synthesis, ligation

dHJ (double-Holliday junction)

 Identification of the double-Holliday junction in vivo
I-SceI
site-specific DSB

2D gel electrophoresis

- In yeast somatic cells, the site-specific DSB generates about 10% as a dHJ
- Among the dHJ formed, 4 times more between sisters than between homologs
Bzymek et al (2010) Nature
The final steps of recombination: resolution or dissolution?

≈ 50-66%

The regulation of this step is not well known in somatic cells

Dissolution by Sgs1-Top3-Rmi1 is the major pathway

 Sgs1/BLM-Top3 mediate dissolution of dHJ

Wu &Hickson (2003) Nature

Cells mutated for the BLM/Sgs1 helicase show a high level of crossovers

Normal Bloom patient

From M. Amor-Gueret, I. Curie

 The resolution of dHJ by resolvases or nucleases

“Canonical” resolvases

major HJ
Very minor
resolution
function
in vivo
in vivo

Wyatt & West (2014) CSHperspectives

The resolution of Holliday Junctions by the canonical resolvases

Wyatt & West (2014) CSHperspectives

 Regulation of homologous recombination

• Regulation of the repair pathway choice by the cell cycle stage

and the chromosomal context
• Homologous recombination and genome stability in cancer cells
• The special case of programmed DSBs
 Regulation of DSB repair pathway depending on the cell
cycle stage
DSB
G1 > G2 S /G2

No sister chromatid
for repair! Repair with the
sister chromatid

- Transcription of CtIP is reduced in the G1 phase

- Phosphorylation of Sae2/CtIP by cyclin-dependent kinases (CDK) ensures that
resection and homologous recombination occur mainly in S and G2 phases

- NHEJ may occur at any cell cycle stage but is predominant in G1 phase (at least
in budding yeast)
- Reduced resection in G1 results from Ku binding to DNA ends, NHEJ, and low
CDK (Cdc28) activity. Elimination of Ku or Dnl4 restores resection initiation to
G1-phase cells, but extensive resection is still partially defective
Phosphorylation of Sae2/CtIP in S/G2 phase activates Mre11
endonuclease
Budding yeast
Human
 Regulation of DSB repair depending on the chromosomal
context

Inducible site-specific DSB

(HR)
(NHEJ)

Aymard et al. (2014) NSMB

 Regulation of DSB repair depending on the chromosomal
context

Ø When a DSB occurs in a transcribed region, CtIP is preferentially recruited and

induces resection and homologous recombination

Aymard et al. (2014) NSMB

 Regulation of DSB repair depending on the nuclear
compartment
A DSB is induced in a sequence in the
nuclear interior or in the heterochromatin
at the nuclear periphery:

Nuclear Nuclear
interior periphery

NHEJ
marker Lamin-associated region

Nuclear pore
HR
marker

Lemaître et al. (2014) Genes Dev

Homologous recombination in repeated regions can lead to
chromosome rearrangements
DSBs occuring in a repeated sequence are at risk of generating
chromosomal rearrangements

duplication
deletion

Guirouilh-Barbat et al. (2014) Frontiers Genet

Homologous recombination in repeated regions can lead to
chromosome rearrangements

translocation

2 direct repeats on the

same chromosome
-> deletion

2 inverted repeats on the

same chromosome
-> inversion

Depending on the
position of centromeres:
-> translocation

-> Dicentric and acentric

chromosomes

Guirouilh-Barbat et al. (2014) Frontiers Genet

 Regulation of DSB repair in repeated regions

Several ways to avoid the rearrangements associated with DSBs occuring in

repeated regions:

- physical exclusion of the broken sequence from its repeated

environnement
- change in the recombination pathway, to favor non homologous end
joining
 Case of the ribosomal DNA (rDNA) repeats in budding yeast

Targeted DSB within the rDNA repeats -> relocation of the DSB outside the nucleolus
Relocation depends on SUMO modification of Rad52
If not relocalized, rDNA hyperrecombination

No DSB
induction

DSB
induction

DSB site nucleolus HR Torres-Rosell et al. (2007) NatCellBiol

DSBs in Drosophila pericentric heterochromatin also move
out to be repaired
• Large stretches of repeated sequences (“satellite repeats”)
• DSB formed in pericentric heterochromatin repeats are resected but sumoylation
blocks HR
• DSB move out to the nuclear periphery to remove SUMO and allow DSB repair by
HR

Ryu et al. (2015) NatCellBiol

 DSBs in mouse pericentric heterochromatin

• Cas9 and guide RNA to target DSB in the major satellite repeats (6 megabases of
234 bp units)

Tsouroula et al. (2016) Mol Cell

 DSBs in mouse pericentric heterochromatin

Ø in G2, DSBs are relocated outside of the repeated regions to be repaired by HR

Tsouroula et al. (2016) Mol Cell

The recombination proteins and diseases

Symington et al (2014) Genetics

 Homologous recombination and genome stability
in cancer cells
When proficient, homologous recombination can produce:

- between repeated, non-allelic sequences: chromosomal

rearrangements: translocations, deletions, inversions that can lead to
tumorigenesis
- between allelic sequences on homologous chromosomes: loss of
heterozygocities (LOH), often seen at the origin of cancers:

Heterozygous Homozygous
mutation mutation
mitotic
2
crossover divisions
homologs

- increased resistance of cancer cells to radiomimetic anti-cancer

drugs (when HR proteins are overexpressed)
 Homologous recombination and genome stability
in cancer cells
• 2 genes, BRCA1 and BRCA2 are frequently mutated in hereditary breast and
ovarian cancers
• Mutation carriers are heterozygous, but tumors often show loss of the WT allele
• They are directly involved in homologous recombination:
CtIP

- BRCA1:
- DSB resection
- RAD51 filament loading

- BRCA2:
- RAD51 filament loading
 BRCA1- or BRCA2-mutated tumors show a high level of mutations
and chromosomal rearrangements
• BRCA1/2-mutated tumors show a high level of genome rearrangements (“LST” Large scale
chromosome translocations) and a high level of mutations (“signature 3”)
 BRCA1- or BRCA2-mutated tumors rely on other
pathways to repair DNA DSBs
(“synthetic lethality”)
BRCA1 and BRCA2-mutated tumors cannot repair DSBs by Homologous Recombination,
even if they are resected
-> rely on both alt-EJ and on NHEJ
HR

NHEJ

Alt-EJ

Highly sensitive to inhibitors of poly(ADP-ribose) polymerases (PARP) = PARPi

-> Currently a therapeutic strategy to treat

BRCA-mutated tumors with PARPi

(Upon PARPi treatment, PARP is trapped at SSBs and at replication forks, which are
transformed into DSB upon replication fork stalling, and also PARP1 is involved in alt-EJ)
 BRCA1- or BRCA2-mutated tumors rely on other
pathways to repair DNA DSBs
(“synthetic lethality”)
- BRCA1 and BRCA2- mutated tumors show synthetic lethality with other mutations:
- Inactivation of the altEJ pathway (Polθ)

-> Currently a therapeutic strategy to treat

BRCA-mutated tumors with Polθ inhibitors

(DNA polymerase)
 In some mutant contexts, BRCA1 deficient tumors
are stable, able to do HR and resistant to
PARPinhibitors

-> useful to assess for these mutant genes in cases of BRCA-mutated tumors resistant to PARPi

Ø Case of 53BP1: its mutation restores resection to BRCA1 mutants

Ø Double mutants BRCA1 53BP1 stable, HR-proficient and resistant to PARPi

Ø (there is enough resection in the double mutants)
 In some mutant contexts, BRCA1 deficient tumors
are stable, able to do HR and resistant to
PARPinhibitors

Ø Case of the shieldin complex: was identified as a mutant that renders BRCA1
mutants resistant to PARPinhibitors

Ø Double mutants of BRCA1 and any member of this complex are stable, HR-
proficient and resistant to PARPi
Ø (there is enough resection in the double mutants)

Greenberg (2018) Nature Cell Biology 20, 862-3.

 Summary for homologous recombination and
cancer

Ø Cancer cells may increase their homologous recombination activity

Ø Resistance to chemotherapeutic agents
Ø Loss of heterozygocities

Ø Homologous recombination is frequently inactivated in cancers (breast, ovary)

Ø Case of Brca1 and Brca2 mutations
Ø These cells show a typical pattern of rearrangements and mutations
Ø These cells are highly sensitive to:
Ø the inhibition of other pathways (alt-EJ)
Ø PARP inhibitors
Ø DNA damaging agents
Ø Many cases of PARPi resistance
Ø Due to mutation of other genes (53BP1 – Shieldin complex)
Ø Important to define other therapeutic targets (pol theta inhibitors…)
 The special case of programmed double-strand breaks

For some developmental programs, DNA double-strand breaks are essential,

although dangerous for genome integrity.

Ø These programmed DSBs are created by dedicated enzymes and associated

partners

Ø Often, the cleavage step is directly coupled to the DSB repair machinery

Ø Programmed Genome Rearrangements in ciliates (PiggyMac enzyme)

Ø V(D)J recombination during the diversification of the immune system (RAG1-

RAG2 enzyme)

Ø Meiotic recombination (Spo11 enzyme)

Review in Betermier, Borde & de Villartay (2020) Trends in Cell Biol

 Programmed Genome Rearrangements in ciliates

Ciliates life cycle

Programmed DNA elimination

gene

45000 sequences must be

precisely eliminated
 Programmed Genome Rearrangements in ciliates
Ø DSBs are introduced by the PiggyMac transposase + 5 additionnal « PiggyMac-like »
non catalytical proteins
 Programmed Genome Rearrangements in ciliates

Ø The DSB repair Ku protein complex is ESSENTIAL for DNA cleavage by the
PiggyMac transposase

Ø Tight coupling between DSB introduction and repair

 V(D)J recombination in the immune system

- Catalyzed by the RAG1/2 enzyme

- DSB repaired by NHEJ, but repair does not absolutely require the Xlf NHEJ factor
 V(D)J recombination in the immune system
- The RAG1/2 complex cleaves and persists on DSB during repair

XRCC4 XLF

Ø 2 redundant synapses ensure proper DSB repair:

- one by XRCC4-XLF (NHEJ)
- the other by RAG1/2 complex
Ø Safeguard mechanism to ensure repair of these programmed DSBs
Programmed DSB formation in meiosis
DSBs

Crossovers
 Programmed DSBs are essential for meiosis

DSBs are essential for correct

chromosome segregation at meiosis I

Random homolog segregation

aneuploid gametes
 Differences between somatic and meiotic DSB repair

Double strand break

Somatic cells Meiotic cells

-> Accidental DSB -> Controlled, programmed DSB

-> Non-Homologous End Joining preferred -> Homologous Recombination preferred

HR: HR:
- Rather with the sister chromatid - Rather with the homologous chromosome
- Preferentially without crossover - Preferentially with crossover
 Many DSBs are formed simultaneously

• 200 (S. cerevisiae) -300 (mammals) programmed DSB/cell

• A fraction is repaired as a crossover

•The programmed DSBs are catalyzed by the Spo11 catalytic subunit +

9 accessory proteins
Spo11 is a homolog of a type II topoisomerase

Covalent link to the 5’

end of DSB
Mre11 complex + Sae2

Homologous
recombination
Spo11 works with several other proteins and interacts
with the Mre11 complex

Mre11
complex
 The Mre11 complex function in meiotic DSB formation

- In budding yeast S. cerevisiae and C. elegans: essential for DSB formation by Spo11
- In mammals: not known because Mre11 complex genes are essential

- In the plant Arabidopsis: not essential, but Mre11 complex interacts with proteins
required for DSB formation

Ø Like for the other programmed DSBs, there is a tight coupling between meiotic DSB
formation and repair, through interactions with the Mre11 complex
Ø May ensure immediate processing of the DSB and their repair by homologous
recombination
 Adaptations of homologous recombination in meiosis

- Programmed DSBs are made by a topoisomerase-like enzyme

Ø need Mre11+Sae2 for repair -> repair only by homologous recombination
- Recombination occurs preferentially with the homolog
Ø need of a meiosis-specific homolog of Rad51, Dmc1 (more stable
filaments)
- A group of meiosis-specific proteins, the « ZMM » stabilize recombination
intermediates
Ø a double-Holliday junction is formed and stabilized
Ø the resolvase that cleaves double-Holliday junctions, Mlh1-Mlh3,
generates only crossovers
Ø ensures enough crossovers are made
 Thank you
Chromosome Dynamics and Recombination lab
Institut Curie, Paris:

- Céline Adam
- Amartya Chakrabouty
- Yulia Gryaznova
- Sophie Loeillet
- Emilie Mylne
- Yoann Nicolas
- Aurore Sanchez