DNA Repair Mechanism – HDR and NHEJ

• Double-strand breaks in DNA are induced by

reactive oxygen species, ionizing radiation, and
chemicals the generate reactive oxygen species
(free radicals).

• Repaired by homologous recombination or

nonhomologous end-joining.
Homologous recombination (Homology
Directed Repair)
• Repairs double-strand breaks by retrieving genetic
information from an undamaged homologous
chromosome.
Nonhomologous end-joining (NHEJ)
• Rejoins double-strand breaks via direct ligation of
the DNA ends without any requirement for
sequence homology.
• Homologous recombination plays a major role in
double-strand break repair in prokaryotes and
single-cell eukaryotes.

• In mammalian cells, double-strand breaks are

primarily repaired through NHEJ.

• In mammalian cells, the main function of

homologous recombination is to repair
double-strand breaks at the replication fork.
Model for mammalian DNA double-strand
break repair by homologous recombination
(Homology Directed Repair)

1. Double-strand break (DSB)

• A DSB is induced by ionizing radiation.

2. End-processing and recognition:

• Recruitment of MRN (Mre11-Rad50-Nbs1) to the

DSB.

• The 3ʹ, 5ʹ exonuclease activity of Mre11 generates

3ʹ ssDNA tails that are recognized by Rad52.
3. Strand invasion and DNA synthesis:

• The 3ʹ tails invade homologous intact sequences.

• Strand exchange generates a hybrid molecule.

• Missing sequence information at the DSB is

restored by DNA synthesis.
4. Branch migration
• Processing of the interlinked molecules.
5. Holliday junction resolution and ligation
ATM activation at double-strand break (DSB)
sites

• ATM (ataxia telangiectasia mutated) is a

serine-threonine kinase

• MRN complexes form a bridge between free DNA

ends via Rad50.

• Inactive ATM is recruited to the DSBs through

interaction with Nsb1.
• ATM is activated by phosphorylation.

• ATM phosphorylates proteins involved in DNA

repair and cell cycle control.

• Patients that lack ATM suffer from a syndrome

called ataxia telangiectasia
– Extreme sensitivity to radiation
– Increased susceptibility to developing cancer
– Immunodeficiency
– Premature aging
– Neurodegenerative disorders
 Holliday junctions

• Early 1960s: Robin Holliday proposed a model for

general recombination based on genetic data
obtained in fungi.

• The model has survived the test of time…

• Heteroduplex DNA: duplex DNA formed during
recombination is composed of single DNA strands
originally derived from different homologs.

• Holliday junction: an intermediate in which the two

recombining duplexes are joined covalently by
single crossovers.
• The Holliday junction is resolved into two
duplexes by an enzyme complex called the
resolvasome.

• The resolvasome has “resolvase” activity.

E. coli RuvABC complex

• Unfolds the Holliday junction arms.

• Uses ATPase activity to promote branch migration.

• RuvC cuts the junction symmetrically in a

sequence-specific manner.
• 2004: The human Holliday junction resolvasome
was purified from 50 liters of HeLa cells passed
through 6 chromatographic steps.

• Rad51C is required for Holliday junction processing

in mammalian cells.

• Rad51C forms a complex with the XRCC3 protein.

Hereditary breast cancer syndromes: mutations
in BRCA1 and BRCA2

• About 5-10% of all cases of breast cancer.

• Mutations in BRCA1 and BRCA2 “tumor suppressor

genes”.

• Lifetime risk for breast (and ovarian) cancer:

BRCA1: 50 to 87%
BRCA2: 15 to 44%
HDR-Gene Correction/Gene Addition
 Nonhomologous end-joining

• This double-strand break repair process can

lead to mutation.

• Two broken ends can be ligated together

regardless of whether they came from the
same chromosome.
 Nonhomologous end-joining

• Frequently results in insertions or deletions

at the break site.

• Trade-off between repair and otherwise

lethal breaks in the genome.
Model for mammalian DNA double-strand
break repair by nonhomologous end-joining

1. Double-strand break
• Induced by ionizing radiation.

2. End recognition
• Broken ends are recognized by heterodimers of
Ku70/Ku80.
3. End processing:
• The endonuclease Artemis is activated by the
DNA-dependent protein kinase catalytic subunit
(DNA-PKCS).

• DNA polymerase (pol) μ or pol λ fill-in gaps and

extend 3ʹ or 5ʹ overhangs.
4. End bridging
• The ligase complex XRCC4-DNA ligase IV is
recruited to the damaged site and forms a bridge.

5. Ligation
• The broken ends are ligated by the XRCC4-DNA
ligase IV complex.
In vitro assays suggest that there is flexibility in
the order of the three key enzymatic steps on
each strand:

•Nucleolytic action

•Polymerization

•Ligation
Non Homologous End Joining
Ku 70/80 heterodimer
NHEJ
 NHEJ Defects

• SCID (Severe Combined Immune Deficiency).

• Cancer – genome instability: rearrangements,
deletions, amplifications.
Homologous Non Homologous
Recombination End Joining
 Genome editing: cleavage repair can either disrupt original
sequence or replace it with a new copy

NEB.com
 Genome editing: cleavage repair can either disrupt original
sequence or replace it with a new copy

“delete”

NEB.com
 Genome editing: cleavage repair can either disrupt original
sequence or replace it with a new copy

“delete” “copy and paste”

NEB.com