Reparación y recombinación I

1. Reparación y recombinación I Evolution could not happen without genetic recombination.

2. Ifitwerenotpossible to exchangematerial between (homologous) chromosomesor genes. • Thecontentofeachchromosomewould be irretrievablyfixed in its particular alleles. • Whenmutationsoccurred, itwouldnot be possible to separate favorable andunfavorablechanges. • Ultimately a chromosomewouldaccumulate so manydeleteriousmutationsthatitwouldfail to function.

3. ¿Dónde ocurre la recombinación? • Recombinationoccursbetweenpreciselycorrespondingsequences.

4. ¿Cuántos tipos de recombinación existen? • Threetypesofrecombinationsharethefeaturethatthe process involvesphysicalexchangeof material between duplex DNAs: • Homologousrecombination • Site-specificrecombinationorSpecializedrecombination • Transposition

5. Recombinación homóloga

6. Recombinación sitio específica

7. Recombinación sitio específica II

8. Recombinationistheresultof: • crossing-overthatoccurs at chiasmataandinvolvestwoofthefourchromatids in meiosis • Crossing-overbetweenanypairofhomologoussecuences in anyphysiologicalconditions

9. Recombinationoccurs by a breakageandreunionthatproceedsviaanintermediateofhybridDNA.

10. Recombination process • A single strands in theregionofthecrossoverexchangetheirpartners. • Thiscreates a stretchofhybridDNA in whichthe single strandofone duplex ispairedwithitscomplementfromtheother duplex.

11. Thekeyevent in recombinationbetweentwo duplex DNAmoleculesisexchangeof single strands. When a single strandfromone duplex displaces itscounterpart in theother duplex, itcreates a branchedstructure. • Theexchangegenerates a stretchofheteroduplexDNAconsistingofonestrandfromeachparent.

12. Two (reciprocal) exchanges are necessary to generate a jointmolecule. • Thejointmoleculeis resolved intotwoseparate duplex molecules by nickingtwooftheconnectingstrands. • Whetherrecombinants are formeddependsonwhetherthestrandsinvolved in the original exchangeortheotherpairofstrands are nickedduringresolution.

13. Branchmigration • Animportantfeatureof a recombinantjointisitsability to movealongthe duplex. • Suchmobilityiscalledbranchmigration. • Thebranchingpoint can migrate in eitherdirection as onestrandis displaced by theother.

14. Branchmigration • Confers a dynamicpropertyonrecombiningstructures. • Thepointofbranchingcannot be established by examining a molecule in vitro (becausethebranch may havemigratedsincethemoleculewasisolated).

15. Branchmigration • Could allowthepointofcrossover in therecombinationintermediate to move in eitherdirection. • Itsrateisuncertain, but in vitro isinadequate to supporttheformationofextensiveregionsofheteroduplexDNA in natural conditions. • Anyextensivebranchmigration in vivo musttherefore be catalyzedby a recombinationenzyme.

16. Hollidaystructure • Thejointmoleculeformed by strandexchangemust be resolved intotwoseparate duplex molecules. • Resolutionrequires a furtherpairofnicks.

17. Hollidaystructure

18. Hollidaystructure • One duplex rotatedrelative to theother. Theoutcomeofthereactiondependsonwhichpairofstrandsisnicked. splicerecombinantDNAmolecules. patchrecombinants.

19. Minimumlength to establishconnectionbetweenrecombiningduplexes • Short homologoussequencescarried by plasmidsorphages are introducedinto bacteria suggestthat therateofrecombinationissubstantiallyreducedifthehomologousregionis<75 bp.

20. Minimumlength to establishconnectionbetweenrecombiningduplexes • Thisdistanceisappreciablylongerthanthe ~10 bprequiredforassociationbetweencomplementary single-strandedregions, • Recombinationimposes demands beyondannealingof complements.

21. Chromosomesmustsynapse (pair) in orderforchiasmata to formwherecrossing-overoccurs. • We can correlatethestagesof meiosis withthe molecular events thathappen to DNA.

22. Recombination • Recombinationisinitiated by making a double-strandbreak in one (recipient) DNA duplex. • Exonucleaseactiongenerates 3’– single-strandedendsthat invade theother (donor) duplex

23. Recombination • NewDNAsynthesisreplacesthe material that has beendegraded. • Thisgenerates a recombinantjointmolecule in whichthetwoDNAduplexes are connected by heteroduplexDNA.

25. Duringtheearlypartof meiosis, homologouschromosomes are paired in thesynaptonemalcomplex. • Each homologue isseparatedfromtheother by a proteinaceouscomplex.

26. Eachchromosomeappears as a mass ofchromatinbounded by a lateral element.

27. Thetripletofparallel dense strandslies in a single planethat curves andtwistsalongitsaxis.

28. Thedistancebetweenthehomologouschromosomesis, more than 200 nm • (diameterofDNAis 2 nm). • A majorproblem in understandingthe role ofthecomplexisthatit do notmakecontactbetweenhomologousDNAmolecules.

29. Proteinsinvolved in synaptonemalcomplexformation • Twogroupsofproteins: • Cohesin • Zip proteins

30. Cohesins • Thecohesinsappear to bind to specificsitesalongthechromosomes in both mitosis and meiosis). • They are likely to play a structural role in chromosomesegregation.

31. Cohesins • Theformationofthe lateral elements may be necessaryforthelaterstagesofrecombination, • Do notpreventtheformationofdouble-strand breaks, theyblockformationofrecombinants.

32. zip1 mutation • Allows lateral elements to formand to becomealigned, butthey do notbecomecloselysynapsed . • TheN-terminal domainof Zip1 proteinislocalized in the central element, buttheC-terminal domainislocalized in the lateral elements.

33. zip1 mutation • Twootherproteins, Zip2 and Zip3 are alsolocalizedwith Zip1. • Thegroupof Zip proteinsformtransverse filaments thatconnectthe lateral elements ofthesisterchromatid pairs.

34. El rompimiento de las dos cadenas es el paso limitante para recombinación • Double-strand breaks thatinitiaterecombinationoccurbeforethesynaptonemalcomplex forms. • Ifrecombinationisblocked, thesynaptonemalcomplexcannotform.

35. Theflushendscreated by thedouble-strandbreak are rapidlyconvertedonbothsidesinto long 3’ single-strandedends. • Mutationof rad50 blockstheconversionoftheflushendintothe single-strandedprotrusionandisdefective in recombination in yeast.

36. Conclusion • Double-strand breaks are necessaryforrecombination

37. Spo11 • In rad50 mutants, the5’ endsofthedouble-strand breaks are connected to theprotein Spo11,homologousto thecatalyticsubunitsof a familyoftypeIItopoisomerases. • Spo11 may be a topoisomerase-likeenzymethatgeneratesthedouble-strand breaks.

38. Spo11 • Spo11 interactsreversiblywithDNA; thebreakisconvertedinto a permanentstructure by aninteractionwithanotherproteinthatdissociatesthe Spo11 complex. • Thenremovalof Spo11 isfollowed by nucleaseaction.

39. Spo11 • At least 9 otherproteins are required tothe process. • Onegroupisrequired to convertthedouble-strand breaks intoprotruding 3’ – OH single-strandedends. • Anothergroupthenenablesthe single-strandedends to invade homologous duplex DNA.

40. Meiosis in S. cerevisiae.

41. Crossovercontrol limitsthenumberofrecombination events betweenmeioticchromosomes to 1-2 crossoversperpairofhomologs. • Mutations in chromosomepairingorsynaptonemalcomplex do notaffecttheother process.

42. Recombination in E. coli • molecular events is similar: • A single strandfrom a broken moleculeinteractswith a partnerduplex • theregionofpairingisextended • andanendonucleaseresolvesthepartnerduplexes. • Enzymesinvolved in eachstage are known, althoughtheyprobablyrepresentonlysomeofthecomponentsrequiredforrecombination.

43. rec–mutations • ThephenotypeofRec–mutantsistheinability to undertakegeneralizedrecombination. • Some~20 loci havebeenidentified.