DNA replication

1. DNAreplication

2. Replicationof duplex DNA • Complexendeavorinvolving a conglomerateofenzymeactivities. • Differentactivities are involved in thestagesofinitiation, elongation, andtermination.

3. Stagesofreplication • Initiationinvolvesrecognitionofanorigin by a complexofproteins. BeforeDNAsynthesisbegins, the parental strandsmust be separatedand (transiently) stabilized in the single-strandedstate. Thensynthesisofdaughterstrands can be initiated at thereplicationfork.

4. Stagesofreplication • Elongationisundertaken by anothercomplexofproteins. • Thereplisomeexistsonly as a proteincomplexassociatedwiththe particular structurethatDNAtakes at thereplicationfork. Itdoesnotexist as anindependentunit (forexample, analogous to theribosome). • As thereplisomemovesalongDNA, the parental strandsunwindanddaughterstrands are synthesized.

5. Stagesofreplication • At theendofthereplicon, joiningand/orterminationreactions are necessary. • Followingtermination, theduplicatechromosomesmust be separatedfromoneanother, whichrequiresmanipulationofhigher-orderDNAstructure.

6. Inability to replicateDNAis fatal for a growingcell. • Mutants in replicationmusttherefore be obtained as conditionallethals. • A comprehensive series ofsuchtemperature-sensitivemutants in E. coliidentifies a set of loci calledthedna genes. • Thednamutantsdistinguishtwostagesofreplication by theirbehaviorwhenthetemperatureisraise.

7. dnamutants • Temperature-sensitive, itcannotsynthesizeDNA at 42°C, but can do so at 37°C.

8. dnamutants • A quick-stop mutant: typeofDNAreplicationtemperature-sensitivemutant (dna) in E. coli thatimmediately stops DNAreplicationwhenthetemperatureisincreased to 42°C.

9. A slow-stopmutantis a typeofDNAreplicationtemperature-sensitivemutant in E. colithat can finish a round ofreplication at theunpermissivetemperature, butcannotstartanother.

10. In vitro complementation • Functionalassayused to identifycomponentsof a process. • Thereactionisreconstructedusing extracts from a mutantcell. • Fractionsfromwild-type cells are thentestedforrestorationofactivity.

11. DNApolymerases • DNAissynthesized in bothsemiconservativereplicationandrepairreactions. • A bacteriumoreukaryoticcell has severaldifferentDNApolymeraseenzymes.

12. DNApolymerases • One bacterial DNApolymeraseundertakessemiconservativereplication; theothers are involved in repairreactions. • Eukaryoticnuclei, mitochondria, andchloroplastseachhave a singleuniqueDNApolymeraserequiredforreplication, andotherDNApolymerasesinvolved in ancillaryorrepairactivities.

13. Hipótesis del mecanismo de Replicación

14. Experimento de Hersey y Chase

16. DNA marcado con 15N y 14N separado por un gradiente de densidad (a)

17. Experimento deMesselsonySthal

18. Experimento deMesselsonySthalII

20. Repairreaction

21. DNA polymerases involved in replication of bacterial and eukaryotic genomes Exonuclease activities Enzyme Subunits 3’-->5’ 5’-->3’ Function Bacterial DNA polymerase DNA polymerase I 1 Yes Yes DNA repair, replication DNA polymerase III At least 10 Yes No Main replicating enzyme Eukaryotic DNA polymerases DNA polymerase a 4 No No Priming during replication DNA polymerase g 2 Yes No Mitochondrial DNA replication DNA polymerase d 2 or 3 Yes No Main replicative enzyme DNA polymerase e At least 1 Yes No Required for detection of DNA damage during genome replication DNA polymerase k 1 or 2? ? ? Required for attachment of cohesin proteins which hold sister chromatids together until the anaphase stage of nuclear division Bacteria and eukaryotes possess other DNA polymerases involved primarily in repair of damaged DNA. These enzymes include DNA polymerases II, IV and V of Escherichia coli and the eukaryotic DNA polymerases b, ζ, η , q and ι. Repair processes are described in Section 14.2.

22. DNA polimerasas I II III pol A 1 103,000 Si aaaaa Si 16-20 aaa 3-200 polB ≥ 4 88,000 Sí aaaaa No ~7 a ≥10,000 polC ≥ 10 ~900,000 Síaaaaaaa No 250-1000aaa ≥500,000 Comparación de las DNA pol de E. coli Gen estructural* Subunidades Mr Exonucleasa 3'5' (corrección de errores) Exonucleasa 5'-->3' Velocidad de polimerización (nucleótidos/s) Procesividad(nucleótidos añadidos antes de disociarse

23. DNA pol de eucariontes • Son al menos 9 polimerasas • Se nombran con letras griegas (igual que las subunidades de la polIII). • La replicasa es laδ • Trabaja en conjunto con el antígeno nuclear de proliferación (PCNA) • PCNA equivale a la subunidad b de la DNApol de E. coli.

24. Processivity • Describes theabilityofanenzyme to performmultiplecatalyticcycleswith a single templateinsteadofdissociatingaftereachcycle.

25. Proofreading (corrección de errores) • Refers to anymechanismforcorrecting errors in proteinornucleicacidsynthesisthatinvolvesscrutinyof individual unitsaftertheyhavebeenadded to thechain.

26. Thefidelityofreplication • Reliesonthespecificityof base pairing. • Errors occurwith a frequencyof ~10-3per base pairreplicated. • The actual rate in bacteria seems to be ~10-8-10-10. • Corresponding to ~1 error pergenomeper 1000 bacterial replicationcycles, or ~10-6per gene pergeneration.

28. Polymerizingandproofreadingactivities • DifferentDNApolymeraseshandletherelationshipbetweenthepolymerizingandproofreadingactivities in differentways. • In some cases, theactivities are partofthesameproteinsubunit, but in othersthey are contained in differentsubunits.

29. Polymerizingandproofreadingactivities • EachDNApolymerase has a characteristic error ratethatisreduced by itsproofreadingactivity. • Proofreadingtypicallydecreasesthe error rate in replicationfrom ~10-5 to ~10-7per base pairreplicated. • Systems thatrecognize errors andcorrectthemfollowingreplicationtheneliminatesomeofthe errors, bringingtheoverallrate to <10-9per base pairreplicated .

30. ReplicaseactivityofDNApoly • Originallydiscovered by a lethalmutation in thednaElocus, whichcodesforthe 130 kDαsubunitthatpossessestheDNAsyntheticactivity.

31. ReplicaseactivityofDNApoly • The 3’ 5’ exonucleolyticproofreadingactivityisfound in anothersubunit, ε, coded by dnaQ. • Thebasic role oftheεsubunit in controllingthefidelityofreplicationin vivoisdemonstrated by theeffectofmutations in dnaQ: thefrequencywithwhichmutationsoccur in the bacterial strainisincreased by >103-fold

32. Aislamiento de diferentesformas de actividadPol III

33. DNA polymerase III holoenzyme

34. DNApolymeraseshave a commonstructure • ManyDNApolymeraseshave a largecleftcomposedofthreedomainsthatresemble a hand. • DNAliesacrossthe "palm" in a groovecreated by the "fingers" and "thumb."

35. DNApolhave a commonstructure • The "palm" domain has importantconservedsequencemotifsthatprovidethecatalytic active site. • The "fingers" are involved in positioningthetemplatecorrectly at the active site. The "thumb" bindstheDNA as itexitstheenzyme, andisimportant in processivity.

36. DNApolhave a commonstructure • Themostimportantconservedregionsofeachofthesethreedomains converge to form a continuoussurface at thecatalyticsite. • Theexonucleaseactivity resides in anindependentdomainwithitsowncatalyticsite.

37. DNApolhave a commonstructure • TheN-terminal domainextendsintothenucleasedomain. DNApolymerasesfallintofivefamiliesbasedonsequencehomologies; thepalmiswellconservedamongthem, butthethumband fingers provideanalogoussecondarystructure elements fromdifferentsequences.

38. Crystalstructureofthe T7 enzymecomplexedwithDNA • Thecatalyticreaction in a DNApolymeraseoccurs at an active site in which a nucleotidetriphosphate pairs withan (unpaired) single strandofDNA.

39. TheDNAliesacrossthepalm in a groovethatiscreated by thethumband fingers. Schematic drawing T7 DNA pol

40. Thumb Fingers Palm

41. Estructura de subunidad tau

42. Replisome • Multiproteinstructurethatassembles at the bacterial replicationfork to undertakesynthesisofDNA. • ItcontainsDNApolymeraseandotherenzymes.

43. Nick translation • Describes theabilityof E. coli DNApolymeraseI to use a nick as a startingpointfromwhichonestrandof a duplex DNA can be degradedandreplaced by resynthesisofnew material; isused to introduce radioactivelylabelednucleotidesintoDNA in vitro.