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Replicacin del DNA

Generalidades, iniciacin en procariontes

MODELS OF DNA REPLICATION


(a) Hypothesis 1:
Semi-conservative replication

(b) Hypothesis 2:
Conservative replication

(c) Hypothesis 3:
Dispersive replication

Intermediate molecule

Meselson and Stahl


Semi-conservative replication of DNA

Isotopes of nitrogen (non-radioactive) were used in this experiment

DNA marcado con 15N y 14N separado por un gradiente de densidad


(a)

Generations 0 HH

0.3 HH

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

0.7

1.0

HL

1.1

HL 1.5

1.9

LL + HL

2.5

3.0

4.1

0 and 1.0 mixed

0 and 4.1 mixed

HL

LL

LL

LH

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 3OH end (primer) In vitro assay for DNA synthesis

Used the assay to purify a DNA polymerizing enzyme DNA polymerase I

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


5 3 5 3 5 3 5 3 3 New progeny strand 3 5 Parental template strand 5 3

5 3

-therefore DNA synthesis occurs only in the 5 to 3 direction

THIS LEADS TO A CONCEPTUAL PROBLEM


Consider one replication fork:
5 Primer

Continuous replication

5
3
Direction of unwinding
5 3

Discontinuous replication
5 3

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

Evidence for Semi-Discontinuous Replication (pulse-chase experiment)

Bacterial culture

Add 3H Thymidine Flood with non-radioactive T Harvest the bacteria at different times For a SHORT time Allow replication after the chase (i.e. seconds) To continue

Bacteria are replicating smallest Isolate their DNA Separate the strands (using alkali conditions) Run on a sizing gradient largest Radioactivity will only be in the DNA that was made during the pulse

Results of pulse-chase experiment


Pulse Chase
5 Primer

smallest

Direction of unwinding
5 3 5 3
largest

*** 5

DNA replication is semi-discontinuous

Continuous synthesis

Discontinuous synthesis

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

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

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

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.

Proof reading activity of the 3 to 5 exonuclease. DNAPI stalls if the incorrect ntd is added - it cant 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.

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

Why the exonuclease activities?


The 5-3' exonuclease activity is used to excise RNA primers in a recation called nick translation

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

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

So if its 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 (CairnsdeLucia 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

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) 1999)

DNAP V: functions in DNA repair (discovered in

DNA Polymerase III


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

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

Replicacin de un cromosoma circular tomando la forma de


(a) (b)

El proceso de la replicacin
Iniciacin. Involucra el reconocimiento de la posicin en dnde empieza la replicacin de una molcula de DNA. Elongacin. Eventos de la horquilla de replicacin en dnde es sintetizada una hebra complementaria. Terminacin. Poco entendia

Inicio de la replicacin en E. coli


El origen de replicacin de E. coli se conoce como oriC. Presenta 245 pb de DNA. Contiene dos motivos cortos repetidos, uno de nueve nucletidos (nonmero), cinco copias y otro de 13 nucletidos (tridecmero) 3 copias.

DnaA, assembles DnaB, DnaC at an open Origin

Bending by HU R-loop formation by RNAP near or at Origin

La protena DnaA se une a las regiones ricas en AT DnaA debe estar acoplada a ATP. 30 molculas de DnaA se unen a oriC La unin ocurre cuando el DNA esta superenrollado negativamente, situacin normal en E. coli.

El resultado de la unin de DnaA a la doble hlice que esta se funde. El mecanismo exacto no se conoce pero parece ser el estrs de torcin inducido por DnaA. La fusin de la hlice es promovida por HU, la protena ms abundante que ayuda a empacar al DNA de E. coli.

Despus de la fusin son reclutadas las protenas 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 regin de hebra sencilla en el origen.

Orgenes de replicacin 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 protenas, el ORC (complejo de reconocimiento del origen). No es precisamente un complejo de iniciacin pues sigue unido despus de iniciada la replicacin.

Estructura del origen de replicacin de levadura

Origen de replicacin en eucariontes superiores


No se ha podido encontrar secuencias ni homlogas ni anlogas a orgenes de replicacin en eucariontes superiores. Podra ser que la replicacin se iniciara en estructuras protenicas que tienen posiciones especficas en el ncleo.

Se describi por Aladjem et al., (1998) una regin de 8 kb que conserv su potencialidad de iniciar la replicacin a alta frecuencia al clonarla en genoma de chimpancs. Protenas con secuencias homlogas a ARSs se han encontrado tambin.

Origin of Replication in SV40


(a mammalian DNA virus)

Paradoxically, Replication is slower in eukaryotes -hence multiple origins

Cis elements in SV40 origin

Multiple sites of initiation in SV40 origin

Viral protein (T-antigen) functions as a helicase

akin to DnaA -which is also a multimer?

SV40 Origin overlaps with a transcriptional promoter

GC elements & SP1 help Replication initiation as well

Elongacin 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

DNA pol en eucariontes y procariontes


Sintetizan polinucletidos en sentido 5-->3 Requiere de un cebador o primer Tienen actividad de exonucleasa 3->5 (no todas)

Sintesis de DNA dependiente de un molde

DNA polymerases involved in replication of bacterial and eukaryotic genomes


Enzyme Subunits Bacterial DNA polymerase DNA polymerase I DNA polymerase III Exonuclease activities 3-->5 5-->3 Function 1 Yes At least 10 Yes Yes No DNA repair, replication Main replicating enzyme

Eukaryotic DNA polymerases DNA DNA DNA DNA polymerase polymerase polymerase polymerase a g d e 4 2 2 or 3 At least 1 1 or 2? No Yes Yes Yes ? No No No No ? Priming during replication Mitochondrial DNA replication Main replicative enzyme Required for detection of DNA damage during genome replication Required for attachment of cohesin proteins which hold sister chromatids together until the anaphase stage of nuclear division

DNA polymerase k

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, Repair processes are described in Section 14.2.

, q and

Comparacin de las DNA pol de E. coli


DNA polimerasas I Gen estructural* Subunidades Mr pol A 1 103,000 II pol B 4 88,000 III pol C 10 ~900,000

Exonucleasa 3->5 (correccin de errores)


Exonucleasa 5'-->3' Velocidad de polimerizacin (nucletidos/s) Procesividad (nucletidos aadidos antes de disociarse

Si aaaaa
Si 16-20 aaa 3-200

S aaaaa
No ~7 a 10,000

Si aaaaa
No 2501000aaa 500,000

Aislamiento de diferentes formas de actividad Pol III


Ce n t ro c atal tic o 2 5 ,0 0 0 e a q 10 ,0 0 0 130,000 Pol III* e q e q Pol III' d g a e q e q Holo e n z im a d b b e q b e q b g a d g Mas a = 9 0 0 ,0 0 0 Co n s i s te d e Po l III' + la s u bu n i dad b (d n a N) S in t e ti z a las h e bras lid e r y re zagad a a d g Mas a = 1 6 5 ,0 0 0 a (p ol C) - S n t e s is de D NA e (d n a Q) - Co rre c c i n 3 '5 ' q - e n s am blaje ? S in t e s is de D NA Mas a = 4 7 0 ,0 0 0 Co n s i s te d e 2 c e n tros + 2 (d n aX )
pro v o c a la di m eri za c i n

In c re m e n t a la p roc e s ivid ad Mas a = 7 5 0 ,0 0 0 Co n s i s te d e Po l II* + c om p le jo gd El c om p le jo gd s e u n e al m o lde

Estructura de subunidad tau

DNA pol de eucariontes


Son al menos 9 polimerasas Se nombran con letras griegas (igual que las subunidades de la pol III). La replicasa es la Trabaja en conjunto con el antgeno nuclear de proliferacin (PCNA) PCNA equivale a la subunidad b de la DNA pol de E. coli.

Cebando la sntesis del DNA

Cebando la sntesis del DNA

Helicasa

Estructura de la helicasa

SSB

Cebando y sintetizando la hebra rezagada

Sntesis en paralelo de la hebra lder y rezagada

Animacin replicacin

Unin de los fragmentos de Okazaki

Endonucleasa FEN1

Dos modelos de acompletar la replicacin resagada

Secuencias de termino en e coli

Problema del primer cebador

Terminacin de la expresnin

Sequences of telomere repeats and telomerase RNAs in various organisms


Species Human Oxytricha Tetrahymena Telomere repeat sequence 5-TTAGGG-3 5-TTTTGGGG-3 5-TTGGGG-3 Telomerase RNA template sequence 5-CUAACCCUAAC-3 5-CAAAACCCCAAAACC-3 5-CAACCCCAA-3

Oxytricha and Tetrahymena are protozoans which are particularly useful for telomere studies because at certain developmental stages their chromosomes break into small fragments, all of which have telomeres: they therefore have many telomeres per cell (Greider, 1996).

Topologa de la terminacin en cromosomas circulares.

Coesinas

Clulas senesentes

Fases del ciclo celular

Cdc6 p

Ciclinas mitticas

Topoisomerasas tipo I y II

Desenrollamiento de la cadena mediante topoisomerasas

Tipos de DNA topoisomerasas

Type

Substrate

Examples

Type IA

Single-stranded DNA

Type IB Type II

Single-stranded DNA Double-stranded DNA

Escherichia coli topoisomerases I and III; yeast and human topoisomerase III; archaeal reverse gyrase Eukaryotic topoisomerase I E. coli topoisomerases II (DNA gyrase) and IV; eukaryotic topoisomerases II and IV

Actividad de topoisomerasas
Las DNA topoisomerasas no desenrollan la doble hlice. Lo que hacen es resolver el problema topolgico contratacando el sobre-enrollamiento que de otra manera tendra la molcula por la progresin de la horquilla de replicacin. El resultado es que la hlice puede ser unzipped poniendo las dos hebras aparte sin que la molcula tenga que rotar.

Modelos de replicacin en DNA circular

Experimento

Experimento de Hersey y Chase II

Inicio de replicacin en E. coli

Sntesis discontinua del DNA

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