Homology directed DSB repair and homologous recombination

Concept behind Homology directed DSB repair

Double-strand breaks (DSBs) are among the most dangerous forms of DNA damage because both DNA strands are severed, meaning there is no intact complementary strand within the same duplex to act as a template for repair.

Homology-directed repair (HDR) solves this problem by using a homologous DNA molecule as a repair template:

• Usually the sister chromatid in late S phase/G2 (high fidelity)

• Occasionally the homologous chromosome in G1 (risk of gene conversion/loss of heterozygosity)

Why HDR Can Repair Without Loss of Information

When the repair template is an identical sister chromatid, the original sequence at the break site is restored exactly.

Therefore:

• No nucleotides are lost

• No sequence is altered

• Repair is essentially error-free

NHEJ

• NHEJ ligates broken ends directly

• Often requires end trimming/alignment via microhomology

• Frequently introduces insertions/deletions

• Predominant in non-dividing/G1/post-mitotic cells

Four Conserved Stages of Homology Directed DSB Repair

HDR proceeds through four mechanistic phases grouped into:

• Presynapsis

• Synapsis

• Postsynapsis

Presynapsis

Processing of the DSB to generate 3' single-stranded DNA tails.

Mechanism

1. Helicases unwind DNA ends

2. Nucleases resect 5' strands preferentially

3. Produces 3' ssDNA overhangs (~50+ nt)

Purpose

• Creates substrate for recombinase loading

• Generates strand capable of homology search and invasion

Synapsis

Pairing of one 3' ssDNA tail with homologous duplex DNA.

Mechanism

1. Recombinase-coated ssDNA searches for homologous duplex

2. Homology recognition (~90–100% identity over 20–30 bp)

3. 3' tail invades duplex

4. D-loop (displacement loop) forms

5. Heteroduplex DNA generated

Postsynapsis

DNA synthesis, strand capture, and restoration of duplex DNA.

Mechanism

1. DNA polymerase extends invading 3' end

2. Repair intermediate processed by either:

   • SDSA pathway

   • Double Holliday junction pathway

3. Remaining gaps filled

4. DNA ligase seals nicks

SDSA Pathway of DSB Repair (Synthesis-Dependent Strand Annealing) mechanism

Step 1: Double-Strand Break

• broken duplex

Step 2: End Resection

• 3’ overhangs generated

Step 3: Strand Invasion / D-loop Formation

• Invading 3' end pairs with homologous template:

  Template duplex opened → D-loop forms

Step 4: DNA Synthesis from Invading 3' End

• DNA polymerase extends invading strand

• Replication bubble migrates along template

Step 5: Newly Synthesised Strand Displaced

• Extended invading strand released from template

Step 6: Annealing to Second Resection End

• Extended strand anneals to complementary second 3' overhang

Step 7: Gap Filling + Ligation

• Remaining ssDNA gaps filled

• Nicks ligated

• Repair complete

SDSA Pathway of DSB Repair (Synthesis-Dependent Strand Annealing) features

• No Holliday junction forms

• No crossover generated

• Major pathway for mitotic DSB repair in eukaryotes

RecBCD Complex in E. coli

Processes DNA ends during presynapsis to generate 3' ssDNA tails.

RecBCD has:

• Helicase activity → unwinds DNA from break

• Nuclease activity → degrades DNA strands

Biological importance:

• Essential for recombinational repair

• recBCD mutants highly DNA damage sensitive

RecBCD Complex in E. coli mechanism

• Binds blunt/broken dsDNA end

• Unwinds and degrades DNA

• Encounters Chi sequence (5'-GCTGGTGG-3')

• Nuclease activity altered

• Preferential degradation of 5' strand

• Leaves 3' ssDNA tail

• Loads RecA onto ssDNA

RecA Protein Function

Bacterial recombinase mediating homology search and strand exchange.

Importance:

• Central catalyst of synapsis

• Required for D-loop formation

• RecA mutants highly recombination defective

Eukaryotic Homologues

• RAD51

• DMC1 (meiosis-specific)

RecA Protein Function mechanism

• Binds ssDNA tail

• Forms helical nucleoprotein filament

• Extends/distorts ssDNA (~50%)

• Searches dsDNA for homology

• Promotes strand invasion

• Catalyses strand exchange

Formation of Double Holliday Junction Recombination Intermediates

Step 1: Initial Strand Invasion

One 3' end invades homologous duplex → D-loop

Step 2: DNA Synthesis

Invading 3' end extended by DNA polymerase

Step 3: Second-End Capture

Second DSB end anneals to displaced D-loop strand

Step 4: Further DNA Synthesis + Ligation

Produces linked duplexes with:

• Two Holliday junctions

Duplex 1 =====X=====X=====

(vertical lines connecting X)

Duplex 2 =====X=====X=====

Holliday Junctions

Four-stranded branched DNA structures linking homologous duplexes during recombination.

Features

• Region of heteroduplex DNA

• Physical linkage of DNA molecules

• Can undergo branch migration

RuvAB Complex Function

Drives branch migration of Holliday junctions in bacteria.

RuvA

• Tetramer binds Holliday junction core

• Opens junction into planar square structure

RuvB

• Hexameric ATP-dependent helicase

• Binds opposite arms of junction

• Pumps DNA through junction

Outcome

• Moves junction along DNA

• Extends/reduces heteroduplex DNA region

RuvC Function

Holliday junction resolvase.

Mechanism

1. Recruited by RuvAB

2. Introduces symmetrical nicks in like-polarity strands

3. Cleaves junction in one of two planes

4. Separates DNA duplexes

Same Plane Resolution of Double Holliday Junctions

Junction 1: Horizontal

Junction 2: Horizontal

Product

• Non-crossover

• Original flanking markers retained

Opposite Plane Resolution of Double Holliday Junctions

Junction 1: Horizontal

Junction 2: Vertical

Product

• Crossover/recombinant

• Exchange of flanking DNA regions

How Resolution Produces Non-Crossover Products

• Both junctions cleaved in same orientation

• DNA arms remain parental configuration

How Resolution Produces Crossover Products

• Junctions cleaved in opposite orientations

• Reciprocal exchange of chromosome arms

Comparison of Homology directed repair of double strand breakages vs homologous recombination

HDR:

• Repair DNA damage

• usually sister chromatid as template

• Sequence restoration/gene conversion

• Holliday junctions usually absent in SDSA

• No crossover

• Mitotic DNA repair

HR:

• exchange genetic information

• Homologous chromosome/other homologous DNA used as template

• Reciprocal exchange possible

• Holliday junctions typically formed

• Often crossover

• Meiosis, gene transfer, recombination

Gene Conversion and Loss of Heterozygosity

• When Homolog Used Instead of Sister Chromatid - Repair copies sequence from homologous chromosome

• Therefore Original allele replaced by homolog allele

• Result

  • Gene conversion

  • Loss of heterozygosity (LOH)

• e.g. Before repair: B / b

  After repair: b / b

• Clinical relevance:

  • Can unmask recessive tumour suppressor mutations

  • Important in cancer progression

Conservation of Recombination Proteins

• DSB Formation - Eukaryotes: Spo11

• End Resection - E.coli: RecBCD, Eukaryotes: MRN Complex

• Recombinase - E.coli: RecA, Eukaryotes: RAD51 / DMC1

• Recombinase loading - E.coli: RecFOR, Eukaryotes: BRCA2 / RAD52

• Branch Migration - E.coli: RuvAB, Eukaryotes: RecQ Family

• Junction Resolution - E.coli: RuvC, Eukaryotes: GEN1