Analysis of the role of Rad5 for the regulation of repair of DSB, small deletions and oxidative damage [Elektronische Ressource] / submitted by Idoia Gómez-Paramio

Idoia Gomez Paramio

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Analysis of the role of Rad5 for the regulation of
repair of DSB, small deletions and oxidative damage












DissertationderFakultätfürBiologie
derLudwigMaximilanUniversitätMünchen











Submittedby
Idoia Gómez Paramio
March7,007














































1. Examiner:+rof.r.ckardtSchupp
2. Examiner:+rof.r.eonhardt
Oralxamination:8.11.2007


IIACKNOWLEDGMENTS


Firstly,Iwouldliketothankmy“Doktormutter”Prof.Dr.FriederikeE ckardtSchupp
forivingehepportunityocarryuthisorksemberferesearchroup.

Iwishtoexpressmysinceregratitudetomyscientificsup ervisorDr.SimoneMörtlfor
her patient guidance, advice and the stimulating discussions, which w ere decisive for the
achievementofthiswork.Herendlessoptimismandenthusiastica ttitudehaveneverceased
toncourageenifficulttimesuringyhesis.

Iowemygratitudetothegroupforcreatingsuchafriendlyatmosp here.EspeciallyI
would like to express my gratitude to Klaudia Winkler for her ext raordinary technical
assistance,butbovell,orernvaluableumannderstanding.lsowishohan karmly
Daniel Sagan for his help but especially for his friendship, fill ing my PhD with enjoyable
moments.

MyspecialthankstoDr.HerbertBrasselmannforteachingme thebasicsofDelphiand
foralltheknowledgeanddedicationnecessarytodevelopGeltool.I alsowouldliketothank
Dr.nnariedloruidingehroughheorldf+FGE.

Finally,mydeepestthankstomyfamilyandfriendsforbringinghappines sintomylife
and in particular to Wolfgang Behr for being at my side, listening to me and giving me
support.






ThisworkwascarriedoutattheInstituteforRadiobiology(ISB)attheGSFForschungszentrumfürUmwelt
undGesundheitandtheFacultyofBiologyattheLudwigMaximiliansUniversity(LMU)inMunich,Germany
duringthetime31.05.200231.12.1005.
ThisstudyhasbeensupportedbythebyEUgrantFIGHCT200200207.
IIIIndex.
1. INTRODUCTION ______________________________________________________ 1
1.1. DNA damage and its repair________________________________________________ 1
1.2. Radiation and its effects __________________________________________________ 1
1.3. DNA Repair ____________________________________________________________ 3
1.4. Direct repair ____________________________________________________________ 4
1.5. Excision repair __________________________________________________________ 4
1.5.1. BaseExcisionRepair __________________________________________________________ 5
1.6. DNA Double Strand Break Repair__________________________________________ 8
1.6.1. HomologousRecombination(HR) ________________________________________________ 8
1.6.2. NonhomologousEndJoining(NHEJ)_____________________________________________ 15
1.6.3. BalancebetweenHRandNHEJ _________________________________________________ 18
1.7. Post-replication repair (PRR)_____________________________________________ 20
1.7.1. Rad5 ______________________________________________________________________ 23
1.8. Goals _________________________________________________________________ 25
2. MATERIALS _________________________________________________________ 27
2.1. Equipment ____________________________________________________________ 27
2.2. Chemicals, Enzyme and other Materials ____________________________________ 28
2.2.1. Chemicals __________________________________________________________________ 28
2.2.2. Enzymes ___________________________________________________________________ 30
2.2.3. Lengthsstandards ____________________________________________________________ 30
2.2.4. Kits _______________________________________________________________________ 30
2.3. Computer programs and web sites_________________________________________ 30
2.4. Oligonucleotides ________________________________________________________ 31
2.5. Plasmids ______________________________________________________________ 33
2.5.1. pJD1 ______________________________________________________________________ 33
2.5.2. pFA6KANMX6______________________________________________________________ 34
2.5.3. pUC19(Fermentas) __________________________________________________________ 35
2.5.4. pGEMT(Promega) __________________________________________________________ 36
2.6. Solutions and buffers ____________________________________________________ 37

2.7. Growth medium ________________________________________________________ 37
2.7.1. Growthmediumforbacteria ____________________________________________________ 37
2.7.2. Growthmediumforyeaststrains ________________________________________________ 37
2.7.3. LiquidHoldingRecoveryBufferforyeast _________________________________________ 38
2.8. Freezing medium for yeast _______________________________________________ 38
2.9. Strains ________________________________________________________________ 39
2.9.1. Bacterialstrain ______________________________________________________________ 39
2.9.2. Yeaststrains ________________________________________________________________ 39
3. METHODS __________________________________________________________ 41
3.1. Microbiology methods ___________________________________________________ 41
3.1.1. Workingwithbacteria_________________________________________________________ 41
3.1.2. Workingwithyeast ___________________________________________________________ 42
3.2. Biomolecular methods ___________________________________________________ 44
3.2.1. IsolationofDNA_____________________________________________________________ 44
3.2.2. CleavageofDNAbyrestrictionendonucleases _____________________________________ 48
3.2.3. SeparationofDNAfragmentsbygelelectrophoresis _________________________________ 49
3.2.4. AmplificationofDNAbyPCR__________________________________________________ 49
3.2.5. PurificationofDNA __________________________________________________________ 50
3.2.6. PhotometricdeterminationofDNAconcentration ___________________________________ 51
IVIndex
3.2.7. SequenceanalysisofDNA _____________________________________________________ 51
3.2.8. Cleavageofthe5’phosphategroups _____________________________________________ 52
3.2.9. LigationofDNArestrictionfragmentsintoplasmids _________________________________ 53
3.2.10. UseofXGALforidentificationofLacZexpression_______________________________ 53
3.3. Determination of the survival capacity after irradiation _______________________ 54
3.3.1. Droptestofyeastcells ________________________________________________________ 54
3.3.2. Survivalcapacityofyeastafterirradiation _________________________________________ 54
3.4. Repair assay with the plasmid pJD1 _______________________________________ 55
3.5. DSB Quantification by Pulsed Field Gel Electrophoresis ______________________ 57
3.5.1. Streaking,irradiationandcultivationofyeastcells __________________________________ 57
3.5.2. DNApreparation_____________________________________________________________ 58
3.5.3. PulsedFieldGelElectrophoresis ________________________________________________ 60
3.5.4. Gelstaininganddensitometryevaluation __________________________________________ 61
3.5.5. QuantificationofDSB_________________________________________________________ 61
3.6. Determination of the mutation capacity ____________________________________ 62
4. RESULTS ____________________________________________________________ 63
4.1. Role of Rad5 in the HR and NHEJ repair pathways __________________________ 63
4.1.1. Generationofrad52mutantsusingthecassetteKANMX6 _____________________________ 63
4.1.2. SurvivalcapacityaftergammaandUVradiation ____________________________________ 64
4.1.3. RoleofRad5fortherepairofchromosomalDSB ___________________________________ 69
4.1.4. RepairofDSBinHRandNHEJdeficientmutants___________________________________ 78
4.1.5. Repairofplasmidialgaps ______________________________________________________ 80
4.2. Role of Rad5 in base excision repair _______________________________________ 88
4.2.1. Generationofknockoutmutants _________________________________________________ 88
4.2.2. Survivalcapacityaftergammairradiation _________________________________________ 92
4.2.3. Repairatchromosomallevel____________________________________________________ 94
4.2.4. SurvivalcapacityafterUVirradiation ____________________________________________ 95
4.2.5. UVmutagenicityinBERmutants _______________________________________________ 96
5. DISCUSSION_________________________________________________________ 99
5.1. Role of Rad5 in the HR and NHEJ repair pathways _________________________ 100
5.1.1. SynergismbetweenRad5andRad52 ____________________________________________ 100
5.1.2. rad5phenotypesuppressioninNHEJdeficientmutants______________________________ 104
5.1.3. Gammainducedrepairdependsonthegrowthphase________________________________ 106
5.2. Role of yKu70 for DSB repair in a HR deficient background __________________ 108
5.3. Haploid strains can repair DSB in high stationary phase _____________________ 110
5.4. Role of Rad5 in the BER repair __________________________________________ 113
5.4