Muller Lecture 3 2008
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Transcript of Muller Lecture 3 2008
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Repair of DS BREAKS
Homology Directed Repair
Non-homology End Joining
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DS Break Repair Pathway
Retrieve Sequenceinformation fromundamaged DNA
Uses recombination Mutants defective in
recombination are UVsensitive (inefficientrepair)
DS DNA BREAKS (or
DSBs)
VERY toxic
Must be dealt with
High Mutagenic
potential
Exploit DSBs for
immunoglobulin
rearrangements
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Origin of DSBOrigin of DSB
Direct DNA Fracture
Replication fork encounters a ssbreak
Daughter strand gap (leading/laggingstrand progression halted by lesion)
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Direct DNA fraction:
Topoisomerase II
cleavages, Ionizing
radiation (minor)
One Ended DSB: Rep
fork encounters a ss DNA
break
Lesion on Template of
daughter strand
DNA replication: High risk of ss break conversion into DSB (very toxic)
Recombinational repair (DSB Repair) CRITICAL to minimizing this risk!!
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DSB REPAIR
Works ONLY when sister chromatid is availableto contribute homology AFTER DNA replication (S-phase)
What about BEFORE DNA Replication (no sisterchromatid available)? Cells use Non-Homologous End Joining or NHEJ
4N2N
S Phase
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- Involves simple end joining. Originally in Eukarya
more recently in Prokaryotes
- No homology but may involve microhomology of a
as few as 1-2 bp.
- Broken ends directly joined
- Misaligned ends may hook up by microhomology
- SS DNA tails snipped off
- Ku protein align ends (highly conserved in bacteria
yeast and man)
- Ku mediated NHEJ pretty messy not efficient
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Core machinery: NHEJ
DSB directly rejoin Ideal for blunt end breaks
Less ideal with non-compatible ends
Microhomology (
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DNA ends 1st bound by Kuheterodimer
Then attracts DNA-PKcs
Forms DNA-PK complex
Then attracts Ligase IVcomplex
SEALED
Eukaryotic NHEJ http://web.mit.edu/engelward-lab/animations/NHEJ.html
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Model for NHEJ
Showing Base related events.
Free DNA ends attract Ku
[protects from degradation]
Ku recruits DNA PK (red)
2 DNA PK subunits
autophoshorylate and phos. Ku
subunits
Phos. DNA PK released
Phos. Ku activates unwindase
Regions of microhomology (short)
hybridize to align
Trim up the whiskers at end
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NHEJ of difficult ends Not all ends readily religatable
Bizarre ends, 5 OH, Phosphates, damaged sugar moities or bases 3Phos. Or 5 OH ends can be processed by polynucleotide kinase
(interacts also with XRCC4)
Artemis nuclease: structure specific can cut hairpins and 3overhangs
Used in V(D)J joining of Immunoglobulin genes
Defects in NHEJ due to Artemis mutation = immunodeficiencysyndrome
WRN Protein has exonuclease activity (mutated in Werners
syndrome)
Eukaryotic NHEJ
DNA repair of DS breaks is foundation of
V(D)J Joining to create antibody diversity
in vertebrates based on NHEJ!
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B cells produce ABs that specifically bind
and recognize a HUGE diversity of
antigens the AB diversity is based on
Recombination and somatic mutation and
clonal selection.
Called
AB composed of 2
copies each L & H
chains.Variable region
defines AG binding
(composed on VLand VH domains
VLVH
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V(D)J Recombination OverviewGenomic Region Light Chain (kappa locus)
Ca. 300 copies of V
gene versions
Any pair of V genes
can fuse with any pair
of J segments
(allows >1200
possible outcomes)
These new segmentsfused to Constant
region by RNA
splicing
Genomic Region Light Chain (kappa locus): Similar to Heavy chain (but H has an additional region called D (for
diversity) which increases diversity (ex: 100 V genes, 12 D genes, 4 J regions = 4800 possible permutations.. The
completed AB can be any pairing of H and L variables. Yields a big number!
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V(D)J Recombination Mechanism
Recombination signal sequences flank the
V(D)J targets.
7mer and 9mer sites Spacer between the 7/9 is either 12 or 23 bp
These bind recombinase
Recombination always occurs between
inverted repeats of 12 bp spacer (one end)and the 23 bp spacer on the other end.
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Recombination Signal
Sequences or RSS
structures
Recombination result with H and Light: Note that they are
flanked with inverted repeats.
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V(D)J Recombination MechanismRecombinase = RAG1, RAG2
RAGs make ss DNA cleavages as
shown & free 3OH attacks opposing
strand to produce a hairpin structureshown
Other Cellular repair proteins (NHEJ
Factors) complete the job,..
Note: its sloppy and involves a few
insertions or additions mutations to
create more diversity.
VERY Similar to transposition processes
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Prokaryotic NHEJ
Recently shown in some bacteria
In Eukarya: Homologous Recombination Recovers DSB
(especially yeast): Limited to S/G2 NHEJ more predominant in higher Eukarya (acts
throughout cell cycle)
Homologues to KU identified in Bacteria (but not
all e.g. enterobacteria like E. coli lack)
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Stars = homologues Ku detected
E. coli
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DSB Repair by NHEJ in Prokaryotes
DS Break forms (Replicative break, Ionizing
Radiation, adduct formation, etc.
Ku Locates site: Serves as end bridging or
alignment factor
Processing enzymes recruited by Ku ringsaround DNA
Ku recedes to all enzyme action (gap filling, exo,
end processing, etc., makes termini suitable for
ligation
Ligation by NHEJ specific ligase
Complex dissociates
NOTE: Prokaryotic Ku is a homodimer whileEukaryotic Ku is heterodimer 70/80
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Ku = homodimer
Ligase: modular with Polymerase, Ligase, nuclease domains.
Qui
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Pseudomonas Ligase Domain structure
Ref: PLOS Genetics 2006 review by Bowater and Doherty
tt ://g ti . l j ur l . rg/ r iv /1553-7404/2/2/ f/10.1371_j ur l. g .0020008-L.
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DS BREAK REPAIR: Homology DrivenDS BREAK REPAIR: Homology Driven
Recombination RepairRecombination Repair
Recover Sequence information by
homology to another region of genome
Accomplished by Finding homologous sequences (best if sister
chromatid)
Tends to be error free/high fidelity (some
exceptions)
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DS DNA Break Repair:
Related to Homologous Rcbn
General comments about Homol. Rcbn. (orHR)
Prokaryotes: HR is RecBCD pathway
Well studied model: See Ch. 10 Watson
DS Breaks (DNA damage) initiate HR
RecA ptn= Binds ss DNA drives pairing & strandinvasion (helps in homology search with SS Binding
protein) RecBCD: Helicase/nuclease process DNA breaks to
generate ss ends for HR invasion
RuvABC: binds Holiday Junctions to resolve
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Resolution
(RuvC)
RecBCD pathway in E. coliDSB somewhere in genome.
Helicase unwinds toward Chi site (every 5KB or so)
Chi: 5GCTGGGTGG
RecA promotes D-loop invasions; helps find homology
Once D-loop hybrids form, RecA/SSB desorb and release
Nick= RecBCD: allows tail to bp
with SS region in other DNA
Gaps/nicks sealed ligase
Branch migration
Resolution gives different
products.
RecA protein: ss DNA
binding ptn that promotes
strand exchange
-Coats ss DNA but not DS
DNA
NOTE: RecA cooperateswith a SSB ptn
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Holiday Junction Resolution Products
Outcomes from Holiday Junction
Cleavages: 2 possible.
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General comments about HR (cont)
Eukaryotes: HR is essential for life
1. Meiosis: Links up homol. Chromosomes for
segregation
2. Recombines parental alleles for offspring
diversity3. Deals with DNA damage: NEXT.
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Homology Directed Repair
Pathways
Synthesis-Dependent Strand Annealing
Classic Double Holiday Junctions: Less evidence
for this in higher eukaryotes
Single Strand Annealing Pathway
Will discuss these two pathways
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Synthesis Dependent Annealing
at DSBs Predominant mechanism Low error rates
Gene Conversion model Mechanistically complex with many
factors will cover from standpoint of
DNA templating process.
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Resection: chewback to 3 overhangs
Binding proteins
mediate (Rad51)
3 nucleofilament seeks
homology elsewhere in
genome
Strand
invasion
forms D loop
DNA Synthesis
across region ofhomology
Sister chromatid: intact wt DNA
Holliday junction
Branch migration releases the
extended strand
Trim
Fill
Ligate
http://web.mit.edu/engelward-lab/animations/SDSA.html
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END Lecture #3
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SS annealing model for repair of DSBsSS annealing model for repair of DSBs
QuickTime 2 and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Works with DNA repeats
(contiguous)
The overhangs (3) simply
anneal
Trim
Ligate
http://web.mit.edu/engelward-lab/animations/SSA.html
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SS annealing model for repair of DSBsSS annealing model for repair of DSBs
Important notes on SS Annealing Model:1. Need adjacent repeats: High
homology important
2. Some sequence loss between repeats
3. One of repeats deleted4. Human genome: lots repeats (Alu
elements x 106, 10% is repeat
sequence anyway)
5. Human genome repeats: highly
polymorphic
6. High sequence diversity in repeats =
reduced efficiency
7. In general: may be a minor pathway
for repair
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Homology Directed Repair or Non-
homologous end Joining?
Which to choose?
1. 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
2. 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.3. 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 (?)
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How to analyze DSB Repair
In human cells: GFP Gene conversion
cassette
Combined with rare cutting restrictionenzymes 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-document&doi=10.1371%2Fjournal.pgen.0030110
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Qui i e and aTIFF (LZW) decompressor
are needed to see this picture.
SceI: rare cutting enzyme (no sites present in huDNA)
Thus-> 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.
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DNA Replication Blockages:
Covalent Adduct
Hairpin
SS nicks
Stably bound
protein (topo)
Other Templating
[transcription shown]
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Enables Replication to Proceed Across DNA Damage or Potentially Blocking Lesions
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TLS
Failsafe backup for lesion misses
Has higher error rate than ideal
TLS still saves the fate of cell fromblockage in DNA replication
Requires specialized D. Pol.
Members of Y polymerases (1999)
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Y Pol properties
N- terminus well conserved cataltyic domain
C-term: less conserved (ptn:ptn interactions forlocalization)
Poorly processive
Synthesis is template dependent but NOT templateddirected Low fidelity (no 3-5 proofreading exonuclease)
Error prone process
All stimulated by PCNA (polymerase sliding clampaccessory ptn.
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TLS polymerases may
incorporate specific NT TLS Not Template Dependent but some ofthe Y pol are specific
Example: DNA Polymerase L Acts at T-T dimers
Tends to insert A residues opposite
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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 (LexArepressor proteolyzed after UV)
Activates the SOS Pathway genes
Includes RecA (recombination protein)
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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
Release of TLS Pol due to
low processivity of
enzyme