Double strand breaks

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Topic 8

• Double strand breaks
• Non-homologous end joining
• Homologous recombination
• Interstrand cross-link repair
• Recombination
• Trans-lesion DNA synthesis

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Repair of double strand breaks (DSBR)

• The repair of double strand breaks, which are
  frequently caused by ionizing radiation but also
  by chemicals, presents a particular challenge
  because the absence of a complementary sequence
  to use as a template makes correct alignment
  difficult.
• Accurate DSBR is extremely important. If such
  breaks are left unrepaired, they may lead to cell
  death but, if repaired improperly, they may lead
  to mutations, rearrangements and chromosome
  translocations that can all initiate neoplastic
  transformation.
• Several methods for DSBR are known including
  homologous recombination and nonhomologous end
  joining. In lower eukaryotes and in bacteria, DSB
  are repaired primarily by homologous
  recombination. This also occurs in mammals but
  many DSBs are repaired in G1 via end joining
  which is more likely to lead to loss or
  alteration of genetic information.

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Phenotype of loss of key proteins in DSBR

• In yeast Many genes were identified by isolating
  radiation sensitive mutants- hence called Rad50
  etc.
• In mice The critical importance of DNA-PK is
  demonstrated by the phenotype of scid mice, which
  have a major defect in DSBR and a defective
  immune system
• Human heritable diseases Ataxia telangiectasia
  and Nijmegen breakage syndrome are autosomal
  recessive conditions in which patients are highly
  sensitive to ionizing radiation, show chromosomal
  instability and a predisposition to cancer.
• Mutants Loss of Ku70 or 80 causes inefficient and
  inaccurate repair. Loss of Rad50, Mre11 or XRCC4
  causes inefficient but mainly accurate repair.
  Loss of DNA ligase IV causes inefficient repair
  with variable accuracy.

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Causes and consequences of double strand breaks

• Many proteins bind to DSBs and may play roles in
  signaling the presence of such damage, protecting
  the ends and causing repair. A critical issue is
  cell cycle arrest which is needed to ensure that
  attempted replication across a double strand
  break does not occur.

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Non-Homologous End-Joining

• NO requirement for DNA sequence similarity or
  complementarity

Requires specialized proteins Ku70/80/DNA-PKcs Ar
temis Fen1 Mre11, Rad50 and NBS XRCC4/DNA ligase
IV
Can be error free but significant chance of DNA
loss or rearrangement
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Non Homologous End Joining
DNA single-strand breaks at MRN-complex may
help in keeping ends together
Ku (Ku70/Ku80) is thought to be the first
protein to bind at each DNA end at such a
double-strand break. Ku functions as a
toolbelt protein to recruit the nuclease,
polymerase, and ligase activities for NHEJ, much
like PCNA or the ß-clamp function during DNA
replication to recruit other activities
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MRN Complex Mre11, Rad50 and Nbs
ATP-controlled DNA-binding processing head
Zinc hook mediated interaction between MRN
complexes on different broken DNA molecules may
result in the tethering and aligning of the DNA
ends
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Non Homologous End Joining
DNA-PK catalytic subunit can bind well to DNA
ends own its own, but its affinity is improved
even further by interaction with Ku. DNA-PKcs
phosphorylates the C-terminus of Artemis The
phosphorylation of the C-terminus alters the
conformation of Artemis such that its C-terminal
tail is no longer inhibitory for the
endonucleolytic activity of Artemis. Artemis can
then function to trim 5' overhangs or 3'
overhangs endonucleolytically
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DNA-dependent protein kinase (DNA-PK)

• DNA-dependent protein kinase (DNA-PK) consists of
  a catalytic subunit and two DNA targeting
  subunits called Ku70 and Ku80. A heterodimer of
  Ku80 and Ku70 binds with high affinity to DNA
  ends protecting them (and possibly positioning
  them) while it recruits and activates the DNA-PK
  catalytic subunit which when activated
  phosphorylates itself, Ku, RPA, XRCC4, p53 and
  H2AX and other proteins.
• Ku then acquires a helicase activity and unwinds
  the DNA ends so that exposed areas of homology
  can anneal.
• The DNA-PK complex has multiple roles which
  include
• (a) prevent transcription/polymerases from trying
  to copy across breaks
• (b) protect ends
• (c) open up ends to allow joining -this needs the
  Artemis protein
• (d) align ends
• (e) provide a scaffolding site for recruiting and
  orienting other factors and the DNA
• (f) be a sensing mechanism saying that ends are
  present.

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Model for DNA-PKcs-Artemis interaction changes in
the presence of a DNA double strand break

• (1). Under cellular salt conditions,
  DNA-PKcs-Artemis cannot bind to DNA ends without
  Ku
• (2). Ku cannot bind to the DNA-PKcsArtemis
  complex without DNA ends
• (3). Ku binding to a DNA end exposes the Ku80 C
  terminus as a docking module for DNA-PKcs, and
  Artemis needs DNA-PKcs for binding to the DNA end
  
• (4). The recruitment of DNA-PKcs to the KuDNA
  complex induces an extensive conformational
  change of DNA-PKcs, and Artemis dissociates under
  kinase-preventive conditions (kinase inhibitor,
  nonactivating DNA end)
• (5). Under kinase-permissive conditions,
  conformational change of partially
  autophosphorylated DNA-PKcs and/or
  phosphorylation of Artemis maintain the
  DNA-PKcs-Artemis association and elicit Artemis
  endonuclease activity.

Drouet, J. et al. J. Biol. Chem.
200628127784-27793
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Non Homologous End Joining
Either DNA polymerase ? or ? can bind to KuDNA
via the BRCT domains within each of these
polymerases These polymerases have specificity
for gapped substrates, and have a lower fidelity
than the X-family pol ?. The XRCC4DNA
ligase IV complex can ligate one strand even if
the other strand is not ligatable. Also the
ligation can occur even when only 2 bp of
annealing exists between the two ends
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Homologous recombinationrepair
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Homologous recombination pathway for break repair

• Requires multiple proteins
• Mre11, Rad50 and Nbs (also involved in NHEJ)
• Rad52
• Rad51B/Rad51C/Rad51D/XRCC2/XRCC3
• RPA
• Rad51

Potentially error free but end processing can
lead to some loss of DNA.
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Rad51- Strand exchange
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Homologous recombination repair
Proposed repair of double-strand breaks
Resolution of Holiday Junction can lead to
crossover of DNA
a b c d
a b c d
a b c d
a b c d
a b c d
c d a b
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Homologous recombination and replication fork
repair.
Leading strand synthesis encounters a DNA
blocking lesion Lagging strand synthesis
continues Fork regression to a Holiday Junction
type structure (Left) Fork progression (Right)
Holiday Junction resolution
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UV induced DNA replication intermediates -
(A) UV-induced lesions are repaired from the
plasmid within 30 min after UV irradiation. (B)
Blocked replication forks and cone region
intermediates transiently accumulate after UV
irradiation. (C) Diagram of the migration
pattern of Pvu II digested pBR322 during 2D
analysis. (D) The replication intermediates
persist until a time correlating with replication
recovery and lesion removal. Replication
recovery, lesion repair, and the relative amount
of replicating fragments (squares) and cone
region intermediates (circles) are plotted.
Replication recovery was assayed by 3Hthymine
incorporation for UV-irradiated (solid symbols)
or mock-irradiated (open symbols) cultures
J. Courcelle et al., Science 299, 1064 -1067
(2003)
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Stalled replication forks
(NER, BER)
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Histone 2AX in response to DNA double-strand
breaks

• a, Double-strand breaks are produced by ionizing
  radiation, chemicals or during the rearrangement
  of immune-receptor genes.
• b, The lesion is detected and various kinase
  enzymes (ATM, ATR and DNA-PK) are activated, in
  turn phosphorylating histone H2AX protein.
• c, ATM, ATR and DNA-PK also transduce the signal
  to downstream proteins (not shown), leading to
  the activation of checkpoints that prevent cell
  division when levels of DNA damage are high, and
  the production of various repair and signalling
  factors, which are recruited to the lesion
• d, Further factors are recruited, aided by the
  phosphorylated H2AX, and perhaps involving
  intermediary proteins.

H2AX may therefore enhance accurate, efficient
repair, or prevent inaccurate or inefficient
repair. H2AX phosphorylation may also lead to
changes in chromatin, or facilitate a checkpoint
that prevents cell division when there is DNA
damage.
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Crosslink Repair

• Replication fork- homologous recombination
• NER with Y-family polymerase

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Cross-link repair

• Model for the mechanism of DNA ICL repair in
  mammalian cells.
• (A) Repair of ICLs is initiated during DNA
  replication.
• (B) The ICL prevents the unwinding of the two DNA
  strands, stalling the replication fork.
• (C) This leads to fork regression and the
  formation of a DSB in an Ercc1-XPF-independent
  manner. The DSB can be detected as a local
  accumulation of H2AX by immunostaining.
• (D) The formation of a DSB creates a substrate
  for the endonuclease Ercc1-XPF in the template
  DNA by revealing a 3' end near the ICL.
• (E) Ercc1-XPF cuts with its characteristic
  substrate specificity (indicated by scissors).
  The incision releases the ICL from one of the two
  DNA strands.

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Cross-link repair

• (F) The residual DNA damage may be bypassed by a
  DNA polymerase capable of translesion synthesis
  (indicated in gold).
• (G) It may be that residual ICL damage is
  ultimately excised from the second strand
  (potential cut sites are indicated with arrows).
• (H) The resulting gap could be filled by the
  replication machinery.
• (I) Repair of the DSB requires resection of the
  broken end to reveal a 3' single-stranded
  overhang.
• (J) This 3' end invades the template DNA to
  create a joint molecule. This is only possible
  once Ercc1-Xpf has incised the blocking ICL. (K)
  Expansion of the heteroduplex could enable
  reestablishment of the replication fork

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Repair of the ICL by excision repair and
translesion DNA synthesis

• (1) Introduction of an ICL into the DNA.
• (2) The NER enzymes UvrABC make incisions in one
  DNA strand both 5' and 3' to the ICL.
• (3) Translesion DNA synthesis is performed by DNA
  polymerase II.
• (4) To excise the cross-linked oligonucleotide,
  UvrABC incises the complementary DNA strand on
  both sides of the ICL.
• (5) The remaining ssDNA gap in the chromosome is
  filled in by DNA polymerase I and ligated,
  resulting in the release of a cross-linked
  oligonucleotide.