1. Cell cycle – Core mechanism
CDK+Cyclin
APC & SCF E3 UB ligases

2. Quiescent cells are found in G0

3. D-type cyclins are highly regulated by external signals
Cyclin D RNA after serum stimulation of starved cells:

4. Restriction point
After CDK/CycD activation and R the cell cycle program becomes cell autonomous and does not respond to external signals
Figure The Biology of Cancer (© Garland Science 2007)

5. Decision about G0/G1 is made during early G1

6. Activation of CycD-CDK leads to phosphorylation and inactivation of RB
Rb phosphorylation releases E2Fs and allows transcription

7. Once G1/S CDK (E-CDK2) is active the positive feedback drives the process forward until completion and return to G1 (APC/C active, Rb active and blocks E2Fs)
Figure The Biology of Cancer (© Garland Science 2007)

8. Start of Cell cycle G1: APC-Hct1(Cdh1) active CDKi present and active
Rb is active and inhibits activity of E2F TFs
Transcription of G1/S,S cyclins is repressed
Activation of G1-CDK causes Rb phosphorylation and activation of E2Fs
Positive feedback loop enhances E2F activity:
E2F activate E2F transcription
G1/S CDK enhances phosphorylation of Rb, and CDKis

9. Feedback loops in the G1-S transition
E2F activates CycE, A and CDK2 - positive FB
CycE/CDK2 inactivates p27 - positive FB
CycA/CDK2 inactivates E2F-
Posphorylates CycE
negative FB
SCF

10. Rb inhibits E2F function
Blocks recruitment of transcription activators

11. Papiloma of the skin

12. Figure 8.35 The Biology of Cancer (© Garland Science 2007)

13. Restriction point
After CDK/CycD activation and R the cell cycle program becomes cell autonomous and does not respond to external signals
Figure The Biology of Cancer (© Garland Science 2007)

14. Replication How is the genome replicated ?
What are origins and how are they assembled ?
3. How are initiation and elongation regulated ?
4. How is re-replication prevented ? 14

15. The human genome is encoded by 3,000,000,000 bp (3x109) which must all be replicated within a few hours with the highest fidelity. The loss of a bp is irreversible and a single mistake could potentially lead to the death of the organism.

16. Analogy One page contains about 3000 letters.
Imagine that you have to copy one million pages in a few hours and have to bind them into 23 volumes.
You can use as many typists as you like but you are allowed no mistakes -no missing letter, no letter twice, and no substitutions.

17. Molecular events in DNA replication
DNA replication is a multi-step process involving:
DNA Polymerase
DNA Helicase (to unwind helix)
Single-Strand Binding Proteins (to stabilize ssDNA)
And more…

18. Table 5-1 Molecular Biology of the Cell (© Garland Science 2008)

19. Processivity is achieved by a sliding clamp
Figure 5-18c Molecular Biology of the Cell (© Garland Science 2008)

20. DNA Sliding clamp
Sliding DNA clamp in bacteria= Beta subunit
The structure of the clamp protein from E.coli as determined by x-ray crystallography, with a DNA helix added to indicate how the protein fits around DNA.
PCNA from Eukaryotes
Sliding clamp form E.coli (Beta subunit)

21. Figure 5-19a Molecular Biology of the Cell (© Garland Science 2008)

22. DNA Sliding clamp PCNA= Proliferating cell nuclear antigen
PCNA is the clamp protein and is expressed specifically in S phase
IHC

23. Origin of replication
DNA replication initiates at defined sites called Origin of replication.

24. Origin of replication DNA replication is bi-directional
Pulse of labeled T

25. The origins of DNA replication on chromosome III of yeast S
The origins of DNA replication on chromosome III of yeast S. cerevisiae (one of smallest eukaryotic chromosomes known)
180 genes
19 replication origins
Green- used in 90% of S phases
Red- used in 10% of S phases
Origins have fixed positions
Not all origins are active in all cells at all times
Not all origins are activated simultaneously
Figure Molecular Biology of the Cell (© Garland Science 2008)

26. An origin of replication in yeast (ARS = Autonomous Replicating Sequence)
5'- T/A T T T A Y R T T T T/A -3’
Origin recognition complex
Components of origins:
ORC binding site
Auxilliary protein binding site (in this case Abf1 facilitates ORC binding)
A stretch of DNA that is easy to unwind (in most origins)
Figure Molecular Biology of the Cell (© Garland Science 2008)

27. Mammalian replication origins
Lack identifiable, genetically required consensus sequence (as opposed to ARS in yeast)
Tend to contain AT-rich sequences, similar to matrix attachment regions (may be similar to yeast)
Nucleotide pools can modulate initiation site selection (Anglana, Cell 114;385, Aug 2003)
1o and 2o origins
Initiation at 1o origin represses initiation at 2o origins
Increasing nucleotide pool reduces frequency of initiation at 2o origins
Reducing nucleotide pool increases frequency at 2o origins
Site-specific initiation is developmentally acquired (change to accommodate changes in gene expression patterns)

28. Parameters controlling site selection
Changes in levels of nucleotide pools
Level of initiation proteins
Chromatin structure
Nuclear organization
DNA methylation

29. Distribution of parental and newly synthesized histones behind a eukaryotic replication fork
Figure 5-38a Molecular Biology of the Cell (© Garland Science 2008)

30. Strategy through which parental patterns of histone H3 and H4 modification can be inherited by daughter chromosomes
Figure Molecular Biology of the Cell (© Garland Science 2008)

31. Mechanism of DNA replication initiation in eukaryotes
Ensures that each origin is activated only once per cell cycle
Figure Molecular Biology of the Cell (© Garland Science 2008)

32. Figure 5–37 Deletions that inactivate an origin of replication in humans. These two deletions are found separately in two individuals who suffer from thalassemia, a disorder caused by the failure to express one or more of the genes in the b-globin gene cluster shown. In both of these deletion mutants, the DNA in this region is replicated by forks that begin at replication origins outside the b-globin gene cluster. The deletion on the left removes DNA sequences that control the chromatin structure of the replication origin on the right.

33. Mammalian replication origins
Lack identifiable, genetically required consensus sequence (as opposed to ARS in yeast)
Tend to contain AT-rich sequences, similar to matrix attachment regions (may be similar to yeast)
Nucleotide pools can modulate initiation site selection (Anglana, Cell 114;385, Aug 2003)
1o and 2o origins
Initiation at 1o origin represses initiation at 2o origins
Increasing nucleotide pool reduces frequency of initiation at 2o origins
Reducing nucleotide pool increases frequency at 2o origins
Site-specific initiation is developmentally acquired (change to accommodate changes in gene expression patterns)

34. Parameters controlling site selection
Changes in levels of nucleotide pools
Level of initiation proteins
Chromatin structure
Nuclear organization
DNA methylation

35. Each eukaryotic chromosome is one linear DNA double helix
Average ~108 base pairs long
DNA replication initiates at many different sites simultaneously.

36. Catenation of sister chromatids is gradually resolved by topoisomerase following replication

37. Distribution of parental and newly synthesized histones behind a eukaryotic replication fork
Figure 5-38a Molecular Biology of the Cell (© Garland Science 2008)

38. Strategy through which parental patterns of histone H3 and H4 modification can be inherited by daughter chromosomes
Figure Molecular Biology of the Cell (© Garland Science 2008)

39. Prevention of re-replication
S-CDKs can not promote replication in G2 cells
Replicated Chromosomes are different from unreplicated chromosome
Replication licensing

40. Only origins loaded with MCM are licensed to replicate
ORC+Cdc6 =clamp loader

41. MCM complex

42. Separating pre-replication from pre-initiating ensures correct replication

43. Pre-replication complex
Pre-initiation complex
S-CDK
S-CDK
S-CDK
Mechanisms to prevent re-replication:

44. Once replication initiates MCM is displaced from the origin and this origin losses license
MCM complex moves along the DNA ahead of the fork and are essential for replication
All CDKs can block replication, defects in CDK activity allow re-replication

45. Figure 17-23 (part 3 of 3) Molecular Biology of the Cell (© Garland Science 2008)

46. Immediately after anaphase, APC/C degrades Geminin and Cyclins and licensing is restored
The Pre-replication complex is reorganized

47. In mammals: Geminin participates in prevention by blocking Cdt1
Geminin Is a substrate for APC/C

48. Figure 17-23 Molecular Biology of the Cell (© Garland Science 2008)

49. Figure 17-22 Molecular Biology of the Cell (© Garland Science 2008)

50. In mammals: Geminin participates in prevention

51. In mammals: Geminin participates in prevention
Geminin Is a substrate for APC/C
Immediately after anaphase, APC/C degrades Geminin and Cyclins and licensing is restored
The Pre-replication complex is reorganized