Hoofdstuk 17: replicatie van DNA foutenherstel
17.1 DNA replicatie
Celcyclus:
• Generatietijd: 18-24h voor sneldelende cellen in cultuur.
• M-fase = mitose + cytokinese (30-45 min in sneldelende cellen). Mitotische index = 3-
5% voor cellen in cultuur.
• S-fase: replicatie (6-8h), gebruik van 3H-deoxythymidine voor meten van lengte van S-
fase
• G1 (variabele duur), eventueel G0 (terminale differentiatie of wachten voor groei-
stimulus)
• G2
DNA synthese komt voor tijdens de S-fase.
DNA replicatie is semi-conservatief.
• Is de replicatie conservatief
of semi-conservatief?
• Meselshon & Stahl: groei
bacteriën, eerst in 15N, dan
14
N medium
• Scheiding zwaar en licht
DNA via densiteits-gradient-
centrifugatie
p.491
DNA replicatie is normaal gezien bidirectioneel.
Prokaryoten:
Theta () replicatie
Vereist ook topoisomerasewerking
, Replicatie gekoppeld aan splitsing
• Ca. 20.000 ORIs (Autonomously Replicating
Sequence in gist, ARS): AT-rijk
• Replicons (replicatie-eenheden), replicatie-
bubbels
Replicatie initieert op gespecialiseerde DNA elementen.
, ORI bindt initiator proteïnen:
- Dna A: ontwinden van 13 meer sequenties
- enkelstrengig-bindingseiwit
- Dna C (helicase) gerecruteerd via Dna B
ORI bindt initiator proteïnen:
- eerst Origin of Recognition Complex (ORC)
- dan MiniChromosome Maintenance (MCM) complex
(helicase) + helicase laders
Pre-replicatie complex: licentie (vergunning) tot replicatie
• Replicatie: 2000 (eukaryoten) – 50.000
(prokaryoten) basen / minuut.
• Lengte van S-fase = bepaald door aantal replicons +
snelheid van activatie (snel in embryonale cellen,
traag in adulte cellen)
• Euchromatine eerder gerepliceerd dan
heterochromatine (5-bromo-deoxyuridine)
DNA polymerasen katalyseren de verlenging van DNA ketens (DNA synthese):
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