Homology Directed Repair

1
Lecture 4

• Homology Directed Repair
• Analysis of Silencing with DSB Repair
  experimental considerations
• Translesion DNA Replication

2
Homology Directed Repair Pathways

• Synthesis-Dependent Strand Annealing
• Single Strand Annealing Pathway

http//web.mit.edu/engelward-lab/animations/SDSA.h
tml
3
SS annealing model for repair of DSBs
http//web.mit.edu/engelward-lab/animations/SSA.ht
ml
Works with DNA repeats (contiguous)
The overhangs (3) simply anneal
Trim Ligate
4
SS annealing model for repair of DSBs

• Important notes on SS Annealing Model
• Need adjacent repeats High homology important
• Some sequence loss between repeats
• One of repeats deleted
• Human genome lots repeats (Alu elements x 106,
  10 is repeat sequence anyway)
• Human genome repeats highly polymorphic
• High sequence diversity in repeats reduced
  efficiency
• In general may be a minor pathway for repair

5
Homology Directed Repair or Non-homologous end
Joining? Which to choose?

• Cell cycle phase Homologous recombination
  requires sister chromatids (limited to S and G2).
  
• Cell Cycle Dependent homology driven repair
• Difficult to perform in bulk chromatin in
  interphase cells
• IF Homologous Repair is NOT suppressed outside of
  S-G2. Mutations will be more frequent as weak
  homology may be selected.
• As diploids can recover sequence off an allele
  but if heterozygous, the parental allele may
  differ.
• Simple, DS breaks with flush ends are rapidly
  re-ligated since the NHEJ pathway is rapid and
  recruited quickly to needed sites. (homology
  directed repair is big and complex slow)
• Difficult breaks may be harder to fix and
  slower to re-ligate (?)

6
Double strand break repair

• Most genotoxic of all DNA damage events
• Repaired by
• Non-homologous end joining minimal templating,
  very error prone
• Homologous Recombination/Gene conversion DNA
  templating recovery from an intact allele by
  synapse and retrieval

What happens to methylation marks when cells
recover genetic information by this
exchange/synthesis mechanism?
7
Post Repair Gene Silencing
8
Gene Silencing Why its important
Cancers have a single prominent
feature Chromosomal Abnormalities (more than one
usually) -Mutation (genetic) -Alterations in gene
regulation
Epigenetic alterations

• Two types
• Stable (male/female specific imprinting)
• Metastable Variable and dynamic (between
  individuals)

9
Cancer cells have growth advantages due to
changes in expression of two groups of growth
control genes

• Tumor Suppressor Genes (TSG)
• Like the brakes on a car
• Tumor Promoting Genes (Oncogenes)
• Like the Accelerator on a car

10
TSG OG

• Negatively Regulate Growth
• Consequences of UP regulation
• Cells stop growing Tissue loss
• Consequences of Dn regulation
• Unregulated growth and Cancer phenotype

• Positively Regulate Growth
• Consequences of UP regulation
• Unregulated growth and Cancer phenotype
• Consequences of Dn regulation
• Cells may cease growing

Epigenetics can alter the balance
11
DNA Methylation

• Epigenetic process of permanent turning off genes
• Mediated by Methylation of Cytosine
• Inappropriate silencing CANCER How does this
  occur mechanistically?
• Link between inappropriate silencing and DNA
  damage

12
Biallelic Inactivation of TSG

• 2 alleles for TSGx (only 1 needed)
• Inactivation of both alleles cancer

Allele 1
Allele 2
13
How is Methylation Regulated?
?
Specifically what could erase methylation marks
and reset silencing in somatic cells? DS Break
repair HR repair
14
How to analyze DSB Repair

• In human cells GFP Gene conversion cassette
• Combined with rare cutting restriction enzymes to
  introduce specific cut sites
• Analysis of gene silencing at repair patch sites
  (methyl-C at CpG sites silenced linked genes).

http//genetics.plosjournals.org/perlserv/?request
get-documentdoi10.13712Fjournal.pgen.0030110
15
SceI rare cutting enzyme (no sites present in
huDNA) Thus-gt transfect cells with SceI gene
construct uniquely cuts at this site to create a
sequence specific DS break Primers allow us to
distinguish Rec and Unrec products at genomic
level GFP signatures Detects gene conversion
event.
16
Treatment with a hypomethylating drug (Aza-dC)
increases GFP positive signatures
GFP Expression Level LOW HIGH
Model
Methyl
Aza
erases methyl gp
Gene Conversion Product (DSB Repaired)
REFERENCE http//genetics.plosjournals.org/p
erlserv/?requestget-documentdoi10.13712Fjourna
l.pgen.0030110
17
Model
Methylated before DNA repair DS BREAK

• Biological Consequences of Recombination-Induced
  Methylation Switch
• Silencing depends on the location of de novo
  methylated CpGs
• Gene off Gene On
• And DNA damage is random therefore HR-induced
  methylation is also random.
• If the expression of the repaired gene is
  lethal, then only cells inheriting the silenced
  copy will survive.
• Conversely, if the function of the repaired gene
  is beneficial or promotes growth, then cells
  inheriting the undermethylated copy will have a
  selective advantage.

De novo methylation mark induced by DNA repair
18
DNA LESIONS How cells can negotiate

• Homologous Recombination
• TLS

19
DNA Replication Blockages
Lesions that block
Covalent Adduct Hairpin SS nicks Stably
bound protein (topo)
DSB Stimulates Recombination
Replication fork
Chicken Foot
E. coli major pathway leading to DSB Repair
Other Templating transcription shown
whats a cell to do?
20
Translesion Synthesis
TLS
Enables Replication to Proceed Across DNA Damage
or Potentially Blocking Lesions
21
TLS

• Failsafe backup for lesion misses
• Has higher error rate than ideal
• TLS still saves the fate of cell from blockage in
  DNA replication
• Requires specialized D. Pol.
• Members of Y polymerases (discovery, 1999)

22
Y Pol properties

• N- terminus well conserved cataltyic domain
• C-term less conserved (ptnptn interactions for
  localization)
• Poorly processive
• Synthesis is template dependent but NOT templated
  directed
• Low fidelity (no 3-5 proofreading exonuclease)
• Error prone process
• All stimulated by PCNA (polymerase sliding clamp
  accessory ptn.)

23
PCNA Eukaryotes

• In eukaryotes, PCNA is a sliding clamp accessory
  protein
• PCNA monoubiquitylated after UV damage and is
  thought to direct PCNA into a TLS mode
• Adding Ubiq. Increases affinity for DNA pol ?
  (TLS pol)
• Ubiquitin added as a result of fork stalling..
  Helicase action continues but DNA new chain
  extension blocked.
• Monoubiquitylation signals TLS pathway
• A deubiquitylating enzyme (isopeptidase, DUB)
  removes monubiquitin, is iteself degrade with UV
  damage.
• PCNA polyubiquitylated and degraded by the
  proteosome pathway (if replication stops due to
  pool depletion (dXTP pools decline with some drug
  treatments).

24
Ubiquitin ub

• Cell tags certain protein with ub
• Can be mono, di, tri or poly-ub
• Polyubiquitylation degradation at 26S
  proteosome complex
• Adding Ubiquitin 3 step process as follows
• E1 ubiq. Activating enzyme transfer ubiquitin
  to next step (ATPase)
• E2 ubiq. Conjugating enzyme Covalent attachment
  of ub to E3 ligase
• E3 ligating enzyme conjugates ub to final
  protein substrate.
• Adding Ub to a protein can
• Alter folding
• Alter sorting
• Induce degradation at proteosome (breakdown)
• Alter biochemical behaviors

25
Example of Ub and TLS mechanism
UV lesion
DNA replication fork collison at UV lesion
off
on
PCNA
Dpol
Ub
TLS
26
Example of Ub and Txn lesion mechanism
UV lesion
Txn complex collison at UV lesion
RPol
ub
Largest catalytic subunit of Rpol if stalled
long enough, will poly-ub and degraded
ub
ub
ub
ub
26S proteosome
27
TLS polymerases may incorporate specific NT

• TLS Not Template Dependent but some of the Y pol
  are specific
• Example DNA Polymerase ?
• Acts at T-T dimers
• Tends to insert A residues opposite

28
TLS in E. coli

• Synthesis directly across lesion
• Complex of UmuC and UmuD
• TLS is so error prone that UmuCD normally not
  present
• SOS Response pathway induces these genes (LexA
  repressor proteolyzed after UV)
• Activates the SOS Pathway genes
• Includes RecA (recombination protein)
• Even in E. coli, proteolytic breakdown is
  important in regulatory controls.

29
TLS DNA SYNTHESIS Pol III Sliding Clamp
encounters TT Dimer Dissociation/fork
stall Translesion DNA Pol inserts bases
opposite dimer Dissociation of TLS Pol DNA
pol III takes over
Polymerase Switch
Release of TLS Pol due to low processivity of
enzyme