Presentation on theme: "Recombination and Repair"— Presentation transcript:
1 Recombination and Repair Chaper 14高雄醫學大學 生物醫學暨環境生物學系張學偉 助理教授
2 Homologous Recombination Concept for chapter 14 & 15Homologous Recombination occur between any two highly similar regions of DNA, regardless of the sequenceNon-homologous (Site-Specific) Recombination (SSR)occur between two defined sequences elements.Transposition (Tn) occur between one specific seq and non-specific DNA sites.
3 Overview of Recombination In all cases of recombination, two DNA molecules are broken and rejoined to each other forming a crossover.Single crossover usually forms short-lived hybrid DNA molecules.promoter recombination of linear chromosomes.cannot cause recombination between two circular DNA molecules.Double crossovers forms recombination.Fig14.1 Two crossovers result in recombination.
7 Crossover due to base homology may occur in DNA as 20-30bases, however, bases is reasonable frequency.Fig Formation of a crossover.
8 heteroduplex:is any region of double-stranded nucleic acid (DNA, RNA), where the two strands come from two different original molecules.
9 Patch recombinants Short parch of heteroduplex remains in each molecule.RuvC, RecG act as resolvase.Formation of two hybrid DNA molecules by crossing-overFig14-4. Rearrangement and Resolution of a Holliday Junction
11 Bind to Junction Drive migration Fig Migration of a Holliday Junction.
12 5 key steps in Homologous recombination (i) alignment of 2 homologous chromosomes(ii) introduction of breaks in DNAs(iii) formation of initial short regions of base pairing betweenthe two recombining DNA molecules (strand invasion)(iv) movement of Holliday junctions by repeat melting andformation of base pair (branch migration)(v) cleavage (or resolution) of Holliday junctions
17 Where is the dsb appeared? Bacterial is haploid. [no HR in sexual reproduction]Bacterial recombination occurs between resident bacterial chrosome and shorter incoming DNA.e.g, transformation, transduction, conjugation.In transformation, a cell can absorb and integrate fragments of DNA from their environment.In conjugation, one cell directly transfers genes (e.g., plasmid) to another cell.In transduction, viruses transfer genes between prokaryotes.
20 Conjugation= plasmid-directed transfer of DNA from one cell to another.
21 Site-specific Recombination (non-homologous recombination) Phage DNA properties is linear inside the virus particleit circularizes upon entering bacterial cells& before integration
22 The control of INT & XIS activity determines it latency or not. att = attachment siteO = center core of 15 bases= the same in phage & bacterialB,P = different in size and sequence inbacterial & phagedsDNAXIS = ExcisionaseINT = integraseThe control of INT & XIS activity determines it latency or not.Fig14-8. Integration of Lambda DNA-overview.
23 Fig14-8. Integration of Lambda DNA-Detail of crossover.
24 Recombination in Higher Organisms resolutionEukaryotic recombination occurs in a span of ~2 hours.Fig Timeline of Eukaryotic Recombination in Yeast.
25 response for recombination and repair Spo11 make dsbRad =response for recombination and repairRad51 ~= RecAFig Spo11 promotes dsb (double strand breaks)
26 Overview of DNA repairDifferent repair enzymes deal with different DNA damages included:Overall distortion of DNA structure. Mismatched RS( more sensitive than ERS) &Excision RSSpecific chemical defects.Lead to mutation.
27 Not included the synthetic enzymes and enzymes also used in normal DNA replication
29 Mismatch Repair Gap filled by DNA Pol III. Cut out part of DNA strand containing wrong base.Mismatch Repair Gap filled by DNA Pol III.Note! most repair system using Pol I to replace short damaged region of DNA.Fig Principle of Mismatch Repair
30 Recognition site is “Sequence-specific” & “Palindromic” Not perturb base pairingDam Protein (product of dam gene) DNA adenine methylaseDcm Protein (product of dcm gene) DNA cytosine methylaseRecognition site is “Sequence-specific” & “Palindromic”GATCCCTGGSequence unique for E.ColiFig Methylated Bases-Chemical Structure.
31 Palindrome make the DNA methylated equally on both strands. Not perturb base pairing[delay in fully methylation]During this period, many repair systems check DNA.Control the initiation of new round of bacterial DNA replicationFunction of methylation Tell which is old, correct strand.Fig Hemimethylated DNA
32 The major mismatch repair system of E.Coli is MutSHL. Consist of MutS, MutH, MutL (proteins)Note! Genes are mutS, mutH, mutL(寫法不一樣)mut = mutator,def in mut high mutation rate
33 Pol III attach & repair the gap created by MutSHL system. H = find the nearest GATC site &nick the non-CH3 strandL = hold togetherFig MutSHL mismatch Repair System
34 General Excision Repair System (“Cut and Patch” Repair) 1. The most widely distributed sysytem for DNA repair.2. Recognize the bulge of DNA strand. e.g., UV (TT dimer)3. Defect UV sensitive (uvr = UV resistence)4. Not detect mismatches, base analogs, certain methylated bases.
35 Pol; 5’exonuclease Fig14-16. UvrABC Excision Repair System Helicase Single strandPol; 5’exonucleaseNick are closed by DNA ligase
36 Excision of Specific Bases (chemical changed bases, CH3, O2) DNA repair byExcision of Specific Bases(chemical changed bases,CH3, O2)deaminationRemoval by DNA glycosylase(- bases)Adenine Hypo-xanthineGuanine XanthineCytosine UracilUracil-N-glycosylase(Ung protein)
37 Pol; 5’exonuclease Pol I 1. recognizes the 3’-OH 2. replaces a strench ofssDNA with AP site.3’-OHPol; 5’exonucleasea-purine/ a-pyrimidineFig Removal of unnatural bases.
38 Prevent incorporation of preformed 8-oxoG into DNA. MutT, MutM, MutYFig dealing with oxidized guanine.
39 Specialized DNA repair mechanisms. 5-methylcytosine leads to mutational hot-spots.Deamination of 5-methylcytosine:G T:G1. Occur spontaneously at any time and rarely during replication.2. Often goes unrepair3. If occur at Dcm recognition site, it is repaired by “ very short patch repair” (Vsr) system [nicking by Vsr endonuclease] Short length of strand remove by DNA pol I
40 O6-CH3-GO4-CH3-TFig Suicide demethylase for O-methyl bases.
41 Note!~CH3 at N- and C- has different effects.Ada = Adaptation to alkylationFig Ada plays a dual role in removing alkyl groups
42 Photoreactivation cleaves thymine dimers PS:Uvr excision repair system also
43 No DNA synthesis 350-500nm photolyase Bind to dimer in dark but lack energy to remove crosslinkFig Photoreactivation cleaves pyrimidine dimers.No DNA synthesis
44 Transcriptional coupling of repair Preferential repair of transcribed template DNA strand.Non-template strand is less likely to be repaired.Bacteria:Transcription-repair coupling factor (TRCF) can detect a stalled RNA pol & direct UrvAB to block site.
45 Fig14-22. Eukaryotic transcription-coupled excision repair. helicaseRecruit the repair proteinNick at the junction between ds and ssDNA.Fig Eukaryotic transcription-coupled excision repair.
47 TT dimer is still unrepaired in this process. Old template is still damaged, but new made is correct.Fig RecA and recombination repair.
48 SOS Error Prone Repair in Bacteria Allow DNA replication to proceed through severely damaged zones, even at the cost of introducing mutations [error prone repair]
49 Fig14-24. RecA and LexA control the SOS system.
50 1.Subunits encoded by umu C and umu D 2. lack of proofreading subunit DNA pol V:1.Subunits encoded by umu C andumu D2. lack of proofreading subunit3. Prefer GA rather than AA to pairTT dimerFor time to repair[no pol activity]Fig DNA polymerase V is part of the SOS system.umu = UV mutagenesis
51 Like E.Coli, yeast, flies, and human all have error-prone DNA polymerase. In higher organisms, these repair enzymes are more specialized and less error-prone.Human error-prone pol, eta, can replicate past TT dimer.
52 Repair in Eukaryotes Human MutS homologue = hMSH2 ~= E.Coli MutS BRCA1 (breast cancer A1) def breast & ovarian ca
53 Double-strand Repair in Eukaryotes byNon-homologous End Joining
54 XRCC4 protein recruits DNA ligase IV to join two broken ends. Fig Non-homologous End Joining in Mammals.
55 Gene conversionNonreciprocal step in DSB-repair sometimes result in gene conversion.Gene conversions are “not” associated with crossing over.Occur at Yeast mating-type switchingat Bacterial genetic exchange via transduction orconjugationat eukaryote homologous recombination in meiosis
56 Fig14-27. Gene Conversion Following Crossing over.
57 Comparison between gene conversion and DNA crossover Comparison between gene conversion and DNA crossover. (a) Two DNA molecules. (b) Gene conversion - the red DNA donates part of its genetic information (e-e' region) to the blue DNA. (c) DNA crossover - the two DNAs exchange part of their genetic information (f-f' and F-F').
58 An origin of gene conversion. (a) Heteroduplexes formed by the resolution of Holliday structure or by other mechanisms. (b) The blue DNA uses the invaded segment (e') as template to "correct" the mismatch, resulting in gene conversion. (c) Both DNA molecules use their original sequences as template to correct the mismatch. Gene conversion does not occur.
59 Fig14-28. Mendelian ratios in Ascospore formation.