Repair of DS BREAKS

Homology Directed Repair

Non-homology End Joining
 DS ‘Break Repair Pathway’
• Retrieve Sequence • DS DNA BREAKS (or
information from DSBs)
undamaged DNA – VERY toxic
• Uses recombination – Must be dealt with
– Mutants defective in – High Mutagenic
recombination are UV
sensitive (inefficient potential
repair) • Exploit DSBs for
immunoglobulin
rearrangements
 Origin of DSB
• Direct DNA Fracture

• Replication fork encounters a ss

break

• Daughter strand gap (leading/lagging

strand progression halted by lesion)
 Lesion on Template of
daughter strand

“One Ended” DSB: Rep

fork encounters a ss DNA
Direct DNA fraction: break
Topoisomerase II
cleavages, Ionizing
radiation (minor)

DNA replication: High risk of ss break conversion into DSB (very toxic)
Recombinational repair (DSB Repair) CRITICAL to minimizing this risk!!
 DSB REPAIR
• Works ONLY when sister chromatid is available
to contribute homology
– AFTER DNA replication (S-phase)

• What about BEFORE DNA Replication (no sister

chromatid available)?
– Cells use Non-Homologous End Joining or NHEJ

S Phase

2N 4N
- 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
Eukaryotic NHEJ

Core machinery: NHEJ

• DSB directly rejoin
– Ideal for blunt end breaks
– Less ideal with non-compatible ends
– Microhomology (<6 bp) important… also error prone
• Machinery at core
– DNA Dependent Protein Kinase (DNA-PK)
– Ligase IV/XRCC4/XLF complex
– Ku70/80 = DNA binding component binds DNA ends as a ‘ring’
– Ku then attracts / activates catalytic subunit DNA-PKcs [ser-thr
kinase
Eukaryotic NHEJ http://web.mit.edu/engelward-lab/animations/NHEJ.html

DNA ends 1st bound by Ku

heterodimer

Then attracts DNA-PKcs

Forms DNA-PK complex

Then attracts Ligase IV

complex

SEALED
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

Eukaryotic NHEJ

NHEJ of difficult ends

• Not all ends readily religatable
– Bizarre ends, 5’ OH, Phosphates, damaged sugar moities or bases
• 3’Phos. Or 5’ OH ends can be processed by polynucleotide kinase
(interacts also with XRCC4)
• Artemis nuclease: structure specific can cut hairpins and 3’
overhangs
– Used in V(D)J joining of Immunoglobulin genes
– Defects in NHEJ due to Artemis mutation = immunodeficiency
syndrome
• WRN Protein has exonuclease activity (mutated in ‘Werner’s
syndrome’)

DNA repair of DS breaks is foundation of

V(D)J Joining to create antibody diversity
in vertebrates… based on NHEJ!
 B cells produce AB’s that specifically bind
and recognize a HUGE diversity of
antigens… the AB diversity is based on
Recombination and somatic mutation and
clonal selection.
Called

te
si
ing VL
ind
b VH
AB composed of 2 Ag
copies each L & H
chains.
Variable region
defines AG binding
(composed on VL
and VH domains
 V(D)J Recombination Overview
Genomic 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 segments

fused 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!
 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.
 Recombination Signal
Sequences or RSS
structures

Recombination result with H and Light: Note that they are

flanked with inverted repeats.
V(D)J Recombination Mechanism
Recombinase = RAG1, RAG2

RAGs make ss DNA cleavages as

shown & free 3’OH attacks opposing
strand to produce a ‘hairpin’ structure
shown

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
 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)
 Stars = homologues Ku detected

E. coli
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

NOTE: Prokaryotic Ku is a homodimer while

Eukaryotic Ku is heterodimer 70/80

Processing enzymes recruited by Ku “rings”

around 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
Ku = homodimer
Ligase: modular with Polymerase, Ligase, nuclease domains.

Pseudomonas Ligase Domain structure

Ref: PLOS Genetics 2006 review by Bowater and Doherty

http://genetics.plosjournals.org/archive/1553-7404/2/2/pdf/10.1371_journal.pgen.0020008-L.pd
QuickTime™ and a
TIFF (Uncompressed) decompressor
f are needed to see this picture.
 DS BREAK REPAIR: Homology Driven
Recombination 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)
 DS DNA Break Repair:
Related to Homologous Rcbn
General comments about Homol. Rcbn. (or
HR)
• 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 & strand
invasion (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
 RecBCD pathway in E. coli
DSB somewhere in genome.
Helicase unwinds toward Chi site (every 5KB or so)
Chi: 5’GCTGGGTGG
RecA promotes D-loop invasions; helps ‘find’ homology

Once D-loop hybrids form, RecA/SSB desorb and release

g
rin
A
Nick= RecBCD: allows tail to bp DN
SS
with SS region in other DNA

D NA
DS
Gaps/nicks sealed ligase
D NA
SS

Branch migration RecA protein: ss DNA

binding ptn that promotes
Resolution strand exchange
(RuvC)
Resolution gives different -Coats ss DNA but not DS
products. DNA
NOTE: RecA cooperates
with a SSB ptn
Outcomes from Holiday Junction
Cleavages: 2 possible.

Holiday Junction Resolution Products

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
diversity
3. Deals with DNA damage: NEXT….
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

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….
 Sister chromatid: intact wt DNA
DNA Synthesis
across region of
homology
Binding proteins
mediate (Rad51)

Holliday junction
Resection: chewback to 3’ overhangs

Branch migration… releases the

extended strand

3’ nucleofilament seeks
homology elsewhere in
genome

Trim

Strand Fill
invasion Ligate
forms D loop

http://web.mit.edu/engelward-lab/animations/SDSA.html
END Lecture #3
SS annealing model for repair of DSBs
http://web.mit.edu/engelward-lab/animations/SSA.html

Works with DNA repeats

(contiguous)

The overhangs (3’) simply

anneal

QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

Trim

Ligate
SS 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 deleted
4. 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
 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 (?)
 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-document&doi=10.1371%2Fjournal.pgen.0030110
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

QuickTime™ and a
Primers allow us to distinguish Rec and (LZW)
TIFF Unrec products
decompressor at genomic level
are needed to see this picture.
GFP signatures: Detects gene conversion event.
 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/perlserv/?request=get-document&doi=10.1371%2Fjournal.pgen.0030110
DNA Replication Blockages:

Covalent Adduct

Hairpin

SS nicks

Stably bound
protein (topo)

Other Templating
[transcription shown]
Enables Replication to Proceed Across DNA Damage or Potentially Blocking Lesions
 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 (1999)
 Y Pol properties
• N- terminus well conserved cataltyic domain
• C-term: less conserved (ptn:ptn 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.
 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
 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)
 TLS DNA SYNTHESIS
Pol III + Sliding Clamp encounters
TT Dimer

Dissociation/fork stall

Translesion DNA Pol inserts

bases opposite dimer
Release of TLS Pol due to
low processivity of
enzyme

Dissociation of TLS Pol

DNA pol III takes over