Mansour Akbari - Host defence_ DSB_11_11_09 - itslearning
Mansour Akbari - Host defence_ DSB_11_11_09 - itslearning
Mansour Akbari - Host defence_ DSB_11_11_09 - itslearning
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
DNA double strand break (<strong>DSB</strong>)DBS is one of the most toxic and mutagenic DNA lesionsCan result in:- loss or rearrangement of genetic information (loss of heterozygosity, translocations)- cell deathSources of <strong>DSB</strong>:Endogenous sources:- reactive oxygen species, collapsed replication, nucleasesExogenous sources:- ionizing radiation, chemicals (e.g. radiomimetic drugs (Bleomycin) used inchemotherapy)
DNA strand break and recombination repair- Intentional strand break and recombination for normal DNA metabolism- Recombination for repair of DNA damage
Recombination: A historical reflectionGregor Mendel(1822-1884)Thomas Hunt Morgan(1866-1945)Uniform F1wing length; eye colorUniform F1P R R P1 : 1 : 1 : 1Principle of independent assortment:Characteristics are inherited independently. I.e.different pairs of alleles are passed to offspringindependently of each other.P R R P1 : 0.1 : 0.1 : 1Linked genes do not assort independently(1910s)
Recombination: Some concepts-Meiosis- Homologous chromosomes- Sister chromatid
Chromosomal crossing-overRecombination takes place between two homologous chromosomes
Chromosomal crossing-overThe outcome of the recombination is that no two offspring of the same parentsare genetically the same (except identical twins).
Cross-strand exchange, the Holliday junction 1964Robin HollidayHe proposed a mechanism of DNA-strand exchangethat occur during meiosis
DNA double strand break (<strong>DSB</strong>)Cells have evolved complex signal-transduction, cell cycle checkpoint and repairpathways to respond to <strong>DSB</strong>Too much overlapping signaling pathways can trigger apoptosis
The major forms of recombinogenic double-strand breaksA: Two-ended DNA double-strand break, created by direct fracture of a DNA duplexB: One-ended DNA double-strand break, created when a replication fork encounters a DNAsingle strand breakC: Daughter strand gap, created when lagging or leading strand progression is inhibited bya DNA lesion.
Major <strong>DSB</strong> repair pathwaysEukaryotic cells repair <strong>DSB</strong>s primarily by two mechanisms;1- homologous recombination (HR)2- non-homologous end-joining (NHEJ)
<strong>DSB</strong> in Mitotic Cells• Replication fork collapseConversion of a SSB into a <strong>DSB</strong> by replicationCleavage of a stalled replication fork• Processing of spontaneous DNA damage• Ionizing radiationCancer treatment; sensitivity studies; cosmic raysResolutionor• Radiomimetic drugs (Bleomycin)Cancer treatment; sensitivity studies
HR, <strong>DSB</strong> end-processingHR starts with extensive 5´ to 3´ end-processingResolutionor
HR, <strong>DSB</strong> end-processingHR starts with extensive 5´ to 3´ end-processingResolutionor
HR, <strong>DSB</strong> end-processing• MRN complex, MRE<strong>11</strong>-RAD50-NBS1• ATP hydrolysisResolutionor• DNA binding by multiple subunits• incision of DNA by its ssDNA endonuclease• 3´to 5´exonuclease activity• tethering of DNA molecules• Exo1• Other exonuclase(s)
<strong>DSB</strong> processing• Possible role in end resection for MRN• ssDNA is found surrounding <strong>DSB</strong>s• Rad50 mutants accumulate unresected <strong>DSB</strong>sResolutionor
ssDNA binding protein RPA and mediatorsssDNARPARad52Resolutionor
RAD51 Nucleoprotein-filamentssDNARPARad52Rad51Resolutionor
RAD51 Nucleoprotein-filamentResolutionor
Strand Exchange (RAD51)• Promotes strand invasion & exchange• Highly conserved in bacteria, yeast, man• Essential in mammals (k/o’s are embryonic lethal)• Required for mitotic and meiotic recombination, <strong>DSB</strong>RResolutionor
Strand Exchange (RAD51)
Strand Exchange (RAD51)DT40 rad51 -/-Resolutionor
HJ processing: the prokaryotic resolvaseRuvCHJ resolvaseResolutionor
Demonstration of HJ processingRuvCImmunoprecipitationHJ *α−XRCC3Pre-Iα−RAD51α−RAD52α−RecAα−RuvCResolution*ResolutionorBeadCX3?
The mammalian resolvase associates with the RAD51 paralogsRAD51BRAD51BCX2DRAD51DXRCC2RAD51CCXRCC3X3ResolutionorStephen C. Y. Ip, Ulrich Ras, Nature 2008Rad2 / XPG familyGEN1 human Yen1 Yeast resolvasesConclusion different mechanisms and proteins are involved
Synthesis dependent strand annealing (SDSA)End resectionStrand invasionFormation of D-loopDNA extensionby POLη and ?Branch migrationRAD54, WRN, BLMFlap processingXPF/ERCC1Repair DNAsynthesis, POLδ
Double-strand break repair via double Holliday junction:
Alternative resolution accounts for the CO/NCO decision
Single-strand annealing (adjacent repeat sequences)
Template switching (lesion bypass)
Replication fork breakdown repair (one-ended <strong>DSB</strong>)
Sister-chromatid exchange:An indicator of DNA damage and cellular DNA repair capacity
Non-homologous end joining (NHEJ)
DNA-end bindingKu70Ku80
DNA-end binding• Binds DNA ends (structure-specific)• Accommodates clean breaks, overhangsas well as hairpins• Caps and protects ends• targeting and binding factor formany NHEJ factors with catalytic activity
DNA-end binding
Non-Homologous-End-joining
DNA-PK csDNA-end bindingDNA-dependent Protein KinaseHoloenzyme
DNA-end binding
Trimming and fill-inMre<strong>11</strong>Rad50Nbs1ArtemisWerner helicasePolymerase (μ, λ, δ?)Polynucleotide kinase
Coordination of the DNA-ends
Coordination of the DNA-ends
End-ligationXRCC4Ligase IV
End-ligationXRCC4XLFLigase IVYeast-Two-Hybrid with XRCC4Structural similarity to XRCC4
End-ligationXRCC4Ligase IVXLF
The core components of NHEJXRCC4Ligase IVXLFCoordinationLigationTrimming• Ku70• Ku80• DNA-PKcsDNA-PK• XRCC4• DNA-LigaseIV• Cernunnos/XLF• Artemis
NHEJ, alternatives
Special uses• Double-Strand-Break Repair in specialized usesHRNHEJMeiotic RecombinationV(D)J recombination
V(D)J recombination for antibody maturationClass switch:From IgM to IgG, IgA
The V(D)J recombination connectionRAG1/RAG2Coding endsSignal endsKuDNA-PKcs:Artemis(DNA-PKcs)KuXRCC4DNA ligase IVXRCC4DNA ligase IVCoding jointSignal joint
How is NHEJ used in <strong>DSB</strong>R?NHEJIt dominates in mammalian cells!HR• This might sound not so attractive – it is generally mutagenic…but most of our DNA is non-coding anyway• Better repair quickly than risk a translocation!
Mis-joining of one-ended DNA <strong>DSB</strong>
<strong>DSB</strong> repair--- by HR or NHEJ ?Main function of HR in eucaryotic mitotic cells is to repair <strong>DSB</strong> cause by arrestand collapse of the DNA replication machinery due to endogenous DNAdamage. NHEJ mostly involved in <strong>DSB</strong>R in non S-phase cells.
Regulation by cell cycle
Additional factorsTemplate accessibilityType of DNA endsRapid binding of Ku proteinsDNA damage singnaling (ATM and MRN)P53 (suppresses HR)
SummaryNon-homologousend-joining (NHEJ)Homologousrecombination (HR)• dominant in G1• activated in S/G2• error-prone (?) • precise (?)• may use microhomologies• depends on large homologies• finds special use in theimmune-system• finds special use in meiosis
Summary•The study of recombination on the level of a population (statistics), and of single events(meiosis in fungi) has a long tradition•This lead to molecular models preceding the actual molecular analysis: a theory driven fieldof research•Today, the original models converge with biochemical, structural biological, and cellularbiological data to give a full picture of HR•Mre<strong>11</strong>-Rad50-NBS1, and the Rad52 epistasis group with the unique strand exchange proteinRad51 are the key players in HR•The original <strong>DSB</strong>R model from 1983 is still valid and has been expanded with models forSDSA and HJ dissolution by BLM/TopoIII
Syndroms associated with deficient repair of <strong>DSB</strong>”Human Chromosome Breakage Syndromes”
RecQ helicase disorders- Belong to the superfamily II group of DNA helicases- Have crucial roles in the maintanace of genome stability- In humans there are five RecQ heilcases. Defects in three RecQ identified
Elevated SCEChrosomal instabilityElevated apoptosis
HR of BLM-dependent resolution
Elevated SCE / HRChrosomal instabilityElevated apoptosisElevated apoptosisChrosomal instability
Elevated SCEChrosomal instabilityElevated apoptosisElevated apoptosisChrosomal instability
Nijmegan breakage syndrome (NBS), mutations in NBS1 and Ataxia-telangiectasia-like disease(ATLD) mutation in MRE<strong>11</strong>, both proteins part of the MRE<strong>11</strong> complex (RAD50, NBS1, MRE<strong>11</strong>)involved in both HR and NHEJ. S-phase check point. Tumours of lymphoid origin, pachygyria.Fanconi anemia. 13 genes (FANC genes) ”E3 mono-ubiquitin ligase core complex (A,B,C,G,E,F, L,M FAAP24) ubiquitinylates the FAND2-FANC1 complex upon stalling of replication forks. FANCMsensor? Exact mechanisms unknown, but important in repair of DNA-crosslinks through HR relatedmechanisms. The “BRACA- FANC” pathway.BRCA1 and BRCA2 (FANCD1),- proteins involved in HR. Mutations in FANCD1—acute myeloidleukemia, microcephaly.BRCA1 loading of RAD51 on RPA coated ssDNA. Mutations in BRCA1 of BRCA2 leads toincreased frequency of carcinoma, e.g. breast and ovarian cancers.
Artemis. Sever Combined Immunodeficiency disroder (SCID)LIG4 syndrome. Immunodeficiency, developmental and growth delay, microcephaly, lymphoidtumors, psorasis, type II diabetes.
Ataxia telangiectasia
Change language