1. Zebrafish Model System
-Production of hundreds of transparent eggs biweekly
-ex utero development of
embryos
-Generation time of 3 months
-Relatively inexpensive
-Vertebrate organism
-Can develop tumors
Day 3

4. The DNA Polymerases Family A Family B Family X Family Y
Pol γ Pol α Pol β Pol η
Pol υ Pol δ Pol λ Pol ι
Pol θ Pol ε Pol μ Pol κ
Pol ζ Pol σ Rev 1

5. Expression Level of Beta

6. Expression level of Eta

7. Expression Level of Iota

8. Expression of Kappa and Rev1

10. Post-Translational Modification of DNA Polymerases
-Ubiquitination – 76 AA, 8.6 kD
-Pol κ and η are both
ubiquitinated which is believed
to be involved in polymerase
switching
-Sumoylation – 11.5 kD
-pol δ has 2 high probability sumoylation sites as predicted
by SUMOplot

13. Regulation of DNA Replication

14. Control of the Cell Cycle
-The proteins that control the cell cycle are different from the proteins that are involved in the process
-Series of checkpoints
-Each checkpoint serves as a biochemical switch

15. Cyclin-Cdk Complexes APC/C Anaphase-Promoting Complex, or Cyclosome
G1-cyclin
APC/C Anaphase-Promoting Complex, or Cyclosome
-Regulated by protein destruction

16. Key Control Components
1. Cyclin-dependent kinases (Cdk)
-Activities vary throughout the cell cycle
-The targets of phosphorylation then varies
2. Cyclins
-Control the activity of Cdk’s
-Undergo synthesis and degradation in
each cell cycle
Cycling of the cyclins creates the cycles of the cyclin/Cdk complex allowing for the varying activities resulting in progression through the cell cycle

17. Control of Proteolysis of APC/C
-Degrading S and M-cyclins stops the Cdk activity and the Cdk’s targets become dephosphorylated and inactive
or Cdh1
or S-cyclin
-APC/C is active in G1 keeping Cdks inactive

18. S phase -Functions -Duplicate the DNA
-Protein packing must be reproduced
-2 issues facing DNA replication
-Accuracy of replication
-Copy DNA only once

19. Control of DNA Duplication
2 steps in Replication
1. Late M/Early G1
-Formation of prereplicative complex (pre-RC)
2. Start of S phase
-Preinitiation complex

20. Initiation of DNA Replication
ORCs are always bound to the origins
pre-RC assembly:
-Inhibited by Cdk
activity
-Stimulated by
APC/C

21. Initiation of DNA Replication
-S-Cdk triggers the preinitiation complex
-ORC and Cdc6 is P by Cdk
-Diassembly of pre-RC
-Cdc6 is degraded
-Cdt1 is inhibited by geminin

22. Initiation of DNA Replication
-DNA Replication is completed
-In late M, APC/C the degradation of geminin
-Cdk activity is decreased, OCRs are dephosphorylated
-Cdc6 is synthesized
-pre-RC assembly

23. Cohesins and Sister Chromatids
-Cohesin – large protein complex which holds the sister chromatids together
Smc (Structural Maintenance of Chromosomes)

24. Sister Chromatid Separation
Securin inhibits separase
Separase cleaves cohesin

25. Next Week
DNA Damage, Repair, and Mutations