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Muller_Lecture__3_2008

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					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




                                        VL
                                   VH
AB composed of 2
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


             Nick= RecBCD: allows tail to bp
             with SS region in other DNA


             Gaps/nicks sealed ligase


             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
                                    TIFF Unrec products
Primers allow us to distinguish Rec and (LZW) 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

				
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