(b) Hypothesis 2:
Conservative replication
(c) Hypothesis 3:
Dispersive replication
Intermediate molecule
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
HL
LL
LL
LH
- dNTPs (substrates) all 4 at once - Mg2+ (cofactor) - ATP (energy source) - free 3OH end (primer) In vitro assay for DNA synthesis
Kornberg also used the in vitro assay to characterize the DNA polymerizing activity
5 3
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)
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
smallest
Direction of unwinding
5 3 5 3
largest
*** 5
Continuous synthesis
Discontinuous synthesis
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.
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
DNAP III: principal DNA replication enzyme DNAP IV: functions in DNA repair (discovered in
1999) 1999)
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).
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
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.
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.
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
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
Si aaaaa
Si 16-20 aaa 3-200
S aaaaa
No ~7 a 10,000
Si aaaaa
No 2501000aaa 500,000
Helicasa
Estructura de la helicasa
SSB
Animacin replicacin
Endonucleasa FEN1
Terminacin de la expresnin
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).
Coesinas
Clulas senesentes
Cdc6 p
Ciclinas mitticas
Topoisomerasas tipo I y II
Type
Substrate
Examples
Type IA
Single-stranded DNA
Type IB Type II
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.
Experimento