Replicación del DNA

1. Replicación del DNA El Replicón

2. Replicon (replicón) • Itis a unitofthegenome in which DNA isreplicated. • Eachrepliconcontainsanoriginforinitiationofreplicationand a terminus.

3. Replicon (replicón) • Theoriginis a sequenceof DNA at whichreplicationisinitiated. • A terminusis a segmentof DNA at whichreplicationends.

4. Control ofreplication • Single copyreplicationdescribes a control system in whichthereisonlyonecopyof a repliconperunitbacterium. • The bacterial andeukarioticchromosomeandsomeplasmidshavethistypeofregulation.

5. Control ofreplication • Multicopy control • Whenthe control systemallowsthereplicon (plasmid) to exist in more thanonecopyper individual bacterial cell.

6. Replicationeye • A is a region in which DNA has beenreplicatedwithin a longer, unreplicatedregion.

7. Replicationfork (horquilla de replicación) • Itisthepoint at whichstrandsof parental duplex DNA are separated so thatreplication can proceed. • A complexofproteinsincluding DNA polymeraseisfound at thefork.

8. Replicationdirection • Unidirectionalreplication • Bidirectionalreplication

9. Origins can be mapped by autoradiographyandelectrophoresis • Replicationforkmovement can be detected by autoradiographyusingradioactive pulses.

10. Bacterial culture *T *T *T *T Grow cells for several generations Small amounts of 3H thymidine are incorporated into new DNA in media with low concentration of 3H- thymidine *T *T *T Add a high concentration of 3H- thymidine Grow for brief period of time *T *T *T *T *T Dense label at the replication fork where new DNA is being made All DNA is lightly labeled with radioactivity *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T John Cairns Cairns then isolated the chromosomes by lysing the cells very very gently and placed them on an electron micrograph (EM) grid which he exposed to X-ray film for two months.

11. Label at both replication forks Evidence points to bidirectional replication

12. T7 DNA replication 1 Replicating bubble in DNA from bacteriophage T7 Two replication forks heading towards opposite ends of the DNA

13. T7 DNA replication 2 B A A’ Replicating DNA from bacteriophage T7. One fork has reached the ends (A and A’). Second fork (arrow) is heading towards the opposite end of the DNA (B)

14. Origins can be mapped by autoradiographyandelectrophoresis • ReplicationforkscreateY-shapedstructures (Horquillas) thatchangetheelectrophoreticmigrationof DNA fragments.

15. The bacterial genomeis a single circular replicon • Bacterial replicons are usuallycirclesthat are replicatedbidirectionallyfrom a single origin. • TheoriginofE. coli, oriC, is 245 bp in length. • Thetworeplicationforksusuallymeethalfway round thecircle, butthere are tersitesthat cause terminationiftheygotoo far.

17. Eacheukaryoticchromosomecontainsmanyreplicons • Eukaryoticreplicons are 40-100 kb in length. • A chromosomeisdividedintomanyreplicons. • Individual replicons are activated at characteristic times during S phase.

19. Nearrepliconsactivate at thesame time Regional activationpatternssuggestthatrepliconsnearoneanother are activated at thesame time. Regions can be characterized as "earlyreplicating" or "late replicating," implyingthatthereislittleinterspersionofrepliconsthatfire at earlyor late times • Visualizationofreplicatingforks by labelingwithDNA precursors identifies 100-300 "foci" insteadofuniformstaining; eachprobablycontains >300 replicationforks. • ThefocicouldrepresentfixedstructuresthroughwhichreplicatingDNAmustmove.

20. Replicationorigins can be isolated in yeast • ARS (autonomousreplicationsequence) isanoriginforreplication in yeast. • Thecommonfeatureamongdifferent ARS sequencesis a conserved 11 bpA•T-richsequencecalledthe A-domain.

21. Replicationorigins can be isolated in yeast • The A domainistheconserved 11 bpsequenceof A-T base pairs in theyeast ARS elementthatcomprisesthereplicationorigin. • The ORC is a complexof 6 proteinsthatbinds to an ARS.

22. ARS Stucture Base compositionratherthansequence may be important in therestoftheregion. Originfunctionisabolishedcompletely by mutations in a 14 bp "core" region, calledthe A domain, thatcontainsan 11 bpconsensussequenceconsistingofA•T base pairs.

23. 7 D loopsmaintainmitochondrialorigins • Mitochondria use differentoriginsequences to initiatereplicationofeach DNA strand. • Replicationofthe H-strandisinitiated in a D-loop. • Replicationofthe L-strandisinitiatedwhenitsoriginisexposed by themovementofthefirstreplicationfork.

24. 7 D loopsmaintainmitochondrialorigins • A D loopis a regionwithinmitochondrial DNA in which a short stretchof RNA ispairedwithonestrandof DNA, displacingthe original partner DNA strand in thisregion. • Thesametermisusedalso to describe thedisplacementof a regionofonestrandof duplex DNA by a complementary single-strandedinvader.

25. Replicationstarts at a specificorigin in the circular duplex DNA. (initiallyonlyoneofthetwo parental strandsisused as a template (the H strand in mammalianmitochondrial DNA) . • Synthesisproceedsforonly a short distance, displacingthe original partner (L) strand, whichremains single-stranded, • Theconditionofthisregiongivesrise to itsname as theDisplacementor D loop.

26. Theendsof linear DNA are a problemforreplication • Specialarrangementsmust be made to replicatethe DNA strandwith a 5′ end.

27. Terminal proteinsenableinitiation at theendsof viral DNAs • Stranddisplacementis a modeofreplicationofsomeviruses in which a new DNA strandgrows by displacingtheprevious (homologous) strandofthe duplex.

28. Adenovirus φ29 replication • Whenthereplicationforkreachestheotherendofthemolecule, the displaced strandisreleased as a free single strand. • Itisthenreplicatedindependently; thisrequirestheformationof a duplex origin by base pairingbetweensome short complementarysequences at theendsofthemolecule.

29. Terminal proteinsenableinitiation at theendsof viral DNAs • A terminal proteinallowsreplicationof a linear phagegenome to start at theveryend. Theproteinattaches to the 5’-endofthegenomethrough a covalentbond, isassociatedwith a DNA polymerase, andcontains a cytosineresiduethatserves as a primer.

30. Terminal proteinsenableinitiation at theendsof viral DNAs • The terminal protein has a dual role: itcarries a cytidinenucleotidethatprovidesthe primer; anditisassociatedwithDNApolymerase. • In fact, linkageof terminal protein to a nucleotideisundertaken by DNApolymerase in thepresenceof adenovirus DNA.

31. Theproteinisfoundcovalentlyattached to each 5’ end. In the case of adenovirus, a terminal proteinislinked to themature viral DNA via a phosphodiesterbond to serine,

32. Rolling circles produce multimersof a replicon • Therollingcircleis a modeofreplication in which a replicationforkproceedsaround a circular templateforanindefinitenumberofrevolutions.

33. Rolling circles produce multimersof a replicon • The DNA strandnewlysynthesized in eachrevolution displaces thestrandsynthesized in thepreviousrevolution, giving a tailcontaining a linear series ofsequencescomplementary to the circular templatestrand.

34. Rollingcircle

36. Rolling circles are used to replicatephagegenom • TheφX A proteinis a cis-actingrelaxasethatgenerates single-strandedcirclesfromthetailproduced by rollingcirclereplication. • A relaxaseisanenzymethat cuts onestrandof DNA, andbinds to the free 5′ end.

37. The A protein has anunusualpropertythat may be connectedwiththeseactivities. Itiscis-acting in vivo. animación

38. The F plasmidistransferred by conjugationbetween bacteria • Conjugationis a process in whichtwo cells come in contactandexchangegenetic material. • In bacteria, DNA istransferredfrom a donor to a recipientcell. In protozoa, DNA passes fromeachcell to theother. A piliis a surfaceappendageon a bacteriumthatallowsthebacterium to attach to other bacterial cells. Itappearslike a short, thin, flexible rod.

39. The F plasmidistransferred by conjugationbetween bacteria • The F plasmidisanepisomethat can be free orintegrated in E. coli, andwhich in eitherform can sponsor conjugation.

40. The F plasmidistransferred by conjugationbetween bacteria • The transfer regionis a segmentonthe F plasmidthatisrequiredfor bacterial conjugation. • Duringconjugation, pili are used to transfer DNAfromonebacterium to another. • Pilinisthesubunitthatispolymerizedintothepilus in bacteria

41. Figura plásmido F A large (~33 kb) regionofthe F plasmid, calledthe transfer region, isrequiredforconjugation. Itcontains ~40 genes that are requiredforthetransmissionof DNA. The genes are named as traandtrbloci. Mostofthem are expressedcoordinately as partof a single 32 kbtranscriptionunit (thetraY-I unit). traMandtraJare expressedseparately. traJis a regulatorthat turns onbothtraMandtraY-I. finPis a regulatorthatcodesfor a smallantisense RNA that turns off traJ.

42. Conjugation transfers single-strandedDNA • WhenanF factor isintegrated, conjugation causes transfer ofthe bacterial chromosomeuntilthe process isinterrupted by (random) breakageofthecontactbetweendonorandrecipient bacteria. • AnHfrcellis a bacteriumthat has anintegratedFplasmidwithinitschromosome. Hfr stands forhighfrequencyrecombination, referring to thefactthatchromosomal genes are transferredfromanHfrcell to anF–cellmuch more frequentlythanfromanF+ cell.

43. Transfer ofanF factor isinitiatedwhenrollingcirclereplicationbegins at oriT. • The free 5’ endinitiates transfer intotherecipientbacterium. • ThetransferredDNAisconvertedintodouble-strandedform in therecipientbacterium. WhenanF factor is free, conjugation "infects" therecipientbacteriumwith a copyoftheF factor.

44. WhenanF factor isintegrated, conjugation causes transfer ofthe bacterial chromosomeuntilthe process isinterrupted by (random) breakageofthecontactbetweendonorandrecipientbac

45. Replicationisconnected to thecellcycle • Bacteria havetwo links betweenreplicationandcellgrowth: • Thefrequencyofinitiationofcyclesofreplicationisadjusted to fittherate at whichthecellisgrowing. • Thecompletionof a replicationcycleisconnectedwithdivisionofthecell.

46. Thereplicationcycle can be defined in terms oftwoconstants: • Cisthefixed time of ~40 minutes required to replicatetheentire bacterial chromosome. • Corresponds to a rateofreplicationforkmovementof ~50,000 bp/minute. • (TherateofDNAsynthesisis more or less invariant at a constanttemperature; itproceeds at thesamespeedunlessanduntilthesupplyof precursors becomeslimiting.)

47. Thereplicationcycle can be defined in terms oftwoconstants: • Disthefixed time of ~20 minutes thatelapsesbetweenthecompletionof a round ofreplicationandthecelldivisionwithwhichitisconnected. • Thisperiod may representthe time required to assemblethecomponentsneededfordivision.

48. Exampleof cells dividingevery 35 minutes. • At division (35/0 minutes), thecellreceives a partiallyreplicatedchromosome.

49. Exampleof cells dividingevery 35 minutes. • Thereplicationforkcontinues to advance. • At 10 minutes, whenthis "old" replicationfork has notyetreachedtheterminus, initiationoccurs at bothoriginsonthepartiallyreplicatedchromosome.

50. Exampleof cells dividingevery 35 minutes. • Thestartofthese "new" replicationforkscreates a multiforkedchromosome.