Replicaci ón del DNA

1. Replicación del DNA Generalidades, iniciación en procariontes

2. MODELS OF DNA REPLICATION (a) Hypothesis 1: (b) Hypothesis 2: (c) Hypothesis 3: Semi-conservative replication Conservative replication Dispersive replication Intermediate molecule

3. Meselson and Stahl Semi-conservative replication of DNA Isotopes of nitrogen (non-radioactive) were used in this experiment

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

5. Generations 0 0.3 0.7 1.0 1.1 1.5 1.9 2.5 3.0 4.1 0 and 1.0 mixed 0 and 4.1 mixed HH HL LL + HL Equilibrium Density Gradient Centrifugation Detection of semiconservative replication in E. coli by density-gradient centrifugation. The position of a band of DNA depends on its content of 14N amd 15N. After 1.0 generation, all the DNA molecules are hybrids containing equal amounts of 14N and 15N HH HL LH LL HL LL

6. Arthur Kornberg (1957) Protein extracts from E. coli + Template DNA Is new DNA synthesized?? - dNTPs (substrates) all 4 at once - Mg2+ (cofactor) - ATP (energy source) - free 3’OH end (primer) In vitro assay for DNA synthesis Used the assay to purify a DNA polymerizing enzyme DNA polymerase I

7. 5’ New progeny strand 3’ 3’ Parental template strand 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ Kornberg also used the in vitro assay to characterize the DNA polymerizing activity - dNTPs are ONLY added to the 3’ end of newly replicating DNA -therefore DNA synthesis occurs only in the 5’ to 3’ direction

8. 3’ 5’ Primer 3’ 5’ Direction of unwinding 3’ Primer Primer 5’ 5’ 3’ 3’ 5’ THIS LEADS TO A CONCEPTUAL PROBLEM Consider one replication fork: Continuous replication Discontinuous replication

9. Evidence for the Semi-Discontinuous replication model was provided by the Okazaki (1968)

10. Flood with non-radioactive T Add 3H Thymidine Bacterial culture Allow replication To continue For a SHORT time (i.e. seconds) Bacteria are replicating smallest largest Evidence for Semi-Discontinuous Replication (pulse-chase experiment) Harvest the bacteria at different times after the chase Isolate their DNA Separate the strands (using alkali conditions) Run on a sizing gradient Radioactivity will only be in the DNA that was made during the pulse

11. Pulse Chase 3’ 5’ Primer 3’ * * * * * * smallest 5’ Direction of unwinding Primer 3’ 5’ 3’ largest Primer 5’ 3’ 5’ Results of pulse-chase experiment

12. DNA replication is semi-discontinuous Continuous synthesis Discontinuous synthesis

13. Features of DNA Replication • DNA replication is semiconservative • Each strand of template DNA is being copied. • DNA replication is bidirectional • Bidirectional replication involves two replication forks, which move in opposite directions • DNA replication is semidiscontinuous • The leading strand copies continuously • The lagging strand copies in segments (Okazaki fragments) which must be joined

14. The Enzymologyof DNA Replication • In 1957, Arthur Kornberg demonstrated the existence of a DNA polymerase - DNA polymerase I • DNA Polymerase I has THREE different enzymatic activities in a single polypeptide: • a 5’ to 3’ DNA polymerizing activity • a 3’ to 5’ exonuclease activity • a 5’ to 3’ exonuclease activity

15. The 5’ to 3’ DNA polymerizing activity Subsequent hydrolysis of PPi drives the reaction forward Nucleotides are added at the 3'-end of the strand

16. Why the exonuclease activities? • The 3'-5' exonuclease activity serves a proofreading function • It removes incorrectly matched bases, so that the polymerase can try again.

17. Proof reading activity of the 3’ to 5’ exonuclease. DNAPI stalls if the incorrect ntd is added - it can’t add the next ntd in the chain Proof reading activity is slow compared to polymerizing activity, but the stalling of DNAP I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizing activity and remove the incorrect base.

18. DNA Replication is Accurate(In E. coli: 1 error/109 -1010 dNTPs added) How? 1) Base-pairing specificity at the active site - correct geometry in the active site occurs only with correctly paired bases BUT the wrong base still gets inserted 1/ 104 -105 dNTPs added 2) Proofreading activity by 3’-5’ exonuclease - removes mispaired dNTPs from 3’ end of DNA - increases the accuracy of replication 102 -103 fold 3) Mismatch repair system - corrects mismatches AFTER DNA replication

19. Why the exonuclease activities? • The 5’-3' exonuclease activity is used to excise RNA primers in a recation called “nick translation”

20. Is DNA Polymerase I the principal replication enzyme?? In 1969 John Cairns and Paula deLucia isolated a mutant bacterial strain with only 1% DNAP I activity (polA) - mutant was super sensitive to UV radiation - but otherwise the mutant was fine i.e. it could divide, so obviously it can replicate its DNA Conclusion: • DNAP I is NOT the principal replication enzyme in E. coli

21. Other clues…. - DNAP I is too slow (600 dNTPs added/minute – would take 100 hrs to replicate genome instead of 40 minutes) - DNAP I is only moderately processive (processivity refers to the number of dNTPs added to a growing DNA chain before the enzyme dissociates from the template) Conclusion: • There must be additional DNA polymerases. • Biochemists purified them from the polA mutant

22. So if it’s not the chief replication enzyme then what does DNAP I do? - functions in multiple processes that require only short lengths of DNA synthesis - has a major role in DNA repair (Cairns- deLucia mutant was UV-sensitive) - its role in DNA replication is to remove primers and fill in the gaps left behind - for this it needs the nick-translation activity

23. The DNA Polymerase Family A total of 5 different DNAPs have been reported in E. coli • DNAP I: functions in repair and replication • DNAP II: functions in DNA repair (proven in 1999) • DNAP III: principal DNA replication enzyme • DNAP IV: functions in DNA repair (discovered in 1999) • DNAP V: functions in DNA repair (discovered in 1999)

24. DNA Polymerase III The "real" replicative polymerase in E. coli • It’s fast: up to 1,000 dNTPs added/sec/enzyme • It’s highly processive: >500,000 dNTPs added before dissociating • It’s accurate: makes 1 error in 107 dNTPs added, with proofreading, this gives a final error rate of 1 in 1010 overall.

25. Iniciación • No es un proceso al azar. • Empieza en una secuencia conocida como origen de replicación • Generalmente se inician dos horquillas de replicación de cada origen (bidireccional). • Un genoma circular bacteriano presenta un solo origen de replicación. • En eucariontes hay multiples orígenes para cada cromosoma. (levadura ~300).

26. Replicación de un cromosoma circular tomando la forma de Θ (b) (a)

27. El proceso de la replicación • Iniciación. Involucra el reconocimiento de la posición en dónde empieza la replicación de una molécula de DNA. • Elongación. Eventos de la horquilla de replicación en dónde es sintetizada una hebra complementaria. • Terminación. Poco entendia

28. Inicio de la replicación en E. coli • El origen de replicación de E. coli se conoce como oriC. • Presenta 245 pb de DNA. • Contiene dos motivos cortos repetidos, uno de nueve nucleótidos (nonámero), cinco copias y otro de 13 nucleótidos (tridecámero) 3 copias.

30. DnaA, assembles DnaB, DnaC at an “open” Origin • Bending by HU • R-loop formation by RNAP near or at Origin

32. La proteína DnaA se une a las regiones ricas en AT • DnaA debe estar acoplada a ATP. • 30 moléculas de DnaA se unen a oriC • La unión ocurre cuando el DNA esta superenrollado negativamente, situación normal en E. coli.

33. El resultado de la unión de DnaA a la doble hélice que esta se funde. • El mecanismo exacto no se conoce pero parece ser el estrés de torción inducido por DnaA. • La fusión de la hélice es promovida por HU, la proteína más abundante que ayuda a empacar al DNA de E. coli.

34. Después de la fusión son reclutadas las proteínas DnaBC, formando el complejo pre-priming. • DnaC tiene un papel transitorio y puede ser que ayude a DnaB a unirse. • DnaB es una helicasa, que puede romper pares de bases. • Incrementa la región de hebra sencilla en el origen.

35. Orígenes de replicación el levadura • Se les llama ARSs (autonomously replicating sequences) • 200 pb • Presenta regiones discretas o subdominios • ORS (secuencia de reconocimiento del origen), 40 pb que es reconocida por un grupo de 6 proteínas, el ORC (complejo de reconocimiento del origen). • No es precisamente un complejo de iniciación pues sigue unido después de iniciada la replicación.

36. Estructura del origen de replicación de levadura

37. Origen de replicación en eucariontes superiores • No se ha podido encontrar secuencias ni homólogas ni análogas a orígenes de replicación en eucariontes superiores. • Podría ser que la replicación se iniciara en estructuras proteínicas que tienen posiciones específicas en el núcleo.

38. Se describió por Aladjem et al., (1998) una región de 8 kb que conservó su potencialidad de iniciar la replicación a alta frecuencia al clonarla en genoma de chimpancés. • Proteínas con secuencias homólogas a ARSs se han encontrado también.

39. Origin of Replication in SV40(a mammalian DNA virus) Paradoxically, Replication is slower in eukaryotes -hence multiple origins

40. Cis elements in SV40 origin

41. Multiple sites of initiation in SV40 origin

42. Viral protein (T-antigen) functions as a helicase akin to DnaA -which is also a multimer?

43. SV40 Origin overlaps with a transcriptional promoter

44. GC elements & SP1 help Replication initiation as well

45. Elongación o alargamiento • Dificultades • Las dos hebras tienen que ser copiadas al mismo tiempo y la polimerasa solo copia de 5’--> 3’ • La hebra retardada debe copiarse de manera discontinua produciendo fragmentos cortos. • La DNA polimerasa necesita cebadores que proporcionen extremos 3’

46. DNA pol en eucariontes y procariontes • Sintetizan polinucleótidos en sentido 5’-->3’ • Requiere de un cebador o primer • Tienen actividad de exonucleasa 3’-->5’ (no todas)

47. Sintesis de DNA dependiente de un molde

48. 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.

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

50. Aislamiento de diferentes formas de actividad Pol III