Download Developmental modifications of the cell cycle involving S phase

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Topic 6
RR: TBA
Chromosomes and the cell cycle
Behavior of chromosomes during a “typical” mitotic cycle:
G1 phase: un-replicated, uncondensed chromosomes
S phase: DNA replication
G2 phase: replicated chromosomes (sister chromatids attached via protein linkages)
M phase: chromosome condensation, nuclear envelope disassembly, chromosomes attach to the
mitotic spindle, chromosome segregation
Cell fusion studies in mammalian cells (1970)- showed that the state of chromatin is regulated
by factors present at different stages of the cell cycle, that promote specific stage-specific
processes. Also showed that factors are present which can prevent inappropriate events from
occurring.
S phase + G1 phase cells fused: S phase cell continues replicating, G1 cell started replicating
DNA. Shows that something normally prevents G1 cells from replicating.
S phase + G2 phase cells fused: S phase cell continues replicating, G2 cell remains quiescent.
Shows that G2 cells are prevented from re-replicating.
S phase + M phase cells fused: S phase cells prematurely enter mitosis and since chromosomes
are not yet completely replicated they cannot do this properly, resulting in “mitotic catastrophe”.
Shows that factors are present in M phase cells that can “push” S phase cells prematurely into M
phase.
1. Identification of cell major cycle regulatory mechanisms
a) Genetic screens (“cdc” mutants) in yeast identified mutations affecting many of the conserved
regulatory factors (blocked at specific stages of the cell cycle)
b) Biochemical studies of MPF (maturation promoting factor) - activity that can induce cells (or
cell-free system) into meiosis or mitosis
Cdks - “Cyclin-dependent kinases”: Different combinations of kinases and cyclins, each
responsible for phosphorylating/regulating ~100s of different proteins. The activation of specific
Cdks is responsible for coordinating specific stages of the cell cycle.
Cdk1: Major regulator of mitosis (Cdk1/CyclinB, Cdk1/CyclinA)
Cdk2: Major regulator of S phase (Cdk2/CyclinE, Cdk2/CyclinA)
Proteosomes- Multi-protein complexes that recognize and degrade modified forms of the major
cell cycle regulatory proteins. Proteosome activity makes progression of the cell cycle
"irreversible", by eliminating proteins required for specific cell cycle events.
Cell cycle "checkpoints" Checkpoints are intrinsic regulatory mechanisms that ensure that cells
have completed previous events in the cell cycle before they proceed to the next stage.
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2. Major regulators of G1/S:
Rb (Retinoblastoma) - blocks replication of S phase genes (transcriptional repressor) and
prevents de-acetylation of origins (blocks necessary step for initiation of S phase).
Cyclin D - complexes with Cdk4/6 kinases, required for inhibition of Rb and activation of Cdk2/E in
response to developmental and nutritional signals, controlling proliferation.
E2F - heterodimeric complex (E2F+DP) acts as transcriptional activator of S phase genes. Also
implicated in acetylation of origins (sites of initiation of S phase).
3. S phase regulation: Initiates with “firing” of replication origins and onset of DNA replication.
Mechanisms that prepare origins for firing normally limit DNA replication to once per cell cycle.
What are replication origins?
a) Yeast origins - ARSs (Autonomously Replicating Sequences)
- defined by plasmid loss experiments (show slide)
- contain 11 bp ACS (ARS Consensus Sequence) plus several less conserved
10-15 bp “B” sequences
-binding site for ORC (Origin Recognition Complex)
b) Metazoan origins - larger, consensus sequence not well defined (probably a
combination of sequence/epigenetic information that is important).
ACE3 (Drosophila chorion gene origin)
The Origin Replication Complex (ORC)
a) “Footprinting” assay used to purify proteins that bind to a yeast ARS (show slide)
b) ORC has 6 constitutive components (ORC1-6), conserved in eukaryotes.
-mutations in ORC genes cause severe DNA replication defects
-mutations in conserved ARS sequences cause defects in ORC binding
c) ORC remains bound to origins throughout the cell cycle. Subunits are phosphorylated
during S/G2/early M phases however, which prevents re-formation of “pre-RC” (see
below).
Where does ORC bind? (S. cerevisiae)
Genome wide ChiP experiment (IP with antibodies against ORC proteins)
- saw ~ 450 sites, most are inter-genic
- 3 factors probably influence origin location (stabilization by ORC/Cdc6
interactions, recruitment by other DNA binding proteins, “open” chromatin
structure - E2F/Rb effect on histone acetylation)
Formation of the pre-Replicative Complex (pre-RC)
During M phase, de-phosphorylation of MCM proteins and re-synthesis of Cdc6 and
Cdt1 allows them to associate with ORC to form a pre-RC, which is maintained until S
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phase initiates. Later, Cdc6 and Cdt1 cooperate to “recruit” DNA replication factors to
the pre-RC, when stimulated by S-Cdk phosphorylation.
MCM proteins (DNA helicases)
a) Genes identified by screens for replication-deficient mutants in mini-chromosome
maintenance assays (show) > loss of ade+ marker carried on plasmid results in “red”
sectored colonies
b) MCMs function as DNA helicases - help to unwind DNA so that DNA polymerase can
engage with the template and synthesize a new strand.
“Ready - Set - Fire”
Once cells receive signals allowing them to enter S phase (E2F “on”/Rb “off”), the pre-RC complex
recruits Cdk2/E (S phase-Cdk) and the DNA replication machinery assembles at the origin (DNA
polymerase, helicases, ssDNA binding proteins etc). “Firing” involves an ATP-dependent
conformational change that activates MCM helicase activity and unwinds the origin. Cdk2/E then
phosphorylates Cdc6, releasing it from ORC (this also targets Cdc6 for SCF-mediated
proteolysis). The replication forks then move away from origins as DNA is synthesized. (show
slide)
Regulation of origin function - “licensing”
a) What prevents origins from re-firing, generating polyploidy cells?
Once S phase begins, Cdk2/E mediated phosphorylation of:
Cdc6 (phosphorylated form is proteolytically degraded, so its not available for pre-RC)
MCM (phosphorylated forms don’t interact with ORC)
ORC proteins (can’t recruit MCM)
M-Cdk (Cdk1/A and Cdk1/B) activity can maintain these modifications, therefore only after mitotic
cyclins are degraded (Cdk1 inactivated) can the mechanism be "re-set".
b) When cell passes through M phase, M-Cdk activity is destroyed by cyclin proteolysis (allowing
phosphatases to de-phosphorylate MCMs and ORC subunits). Mitosis also allows re-synthesis of
Cdc6, de-phosphorylation of Mcms, and ORCs. This mechanism is called "licensing" because it
ensures cells only replicate once per cell cycle.
Developmental modifications of the cell cycle involving S phase
a) Meiosis - S phase omitted between MI and MII
c) Endoreplication cycles - successive S phases, separated by gap phases (alternate mechanisms
used to inactivate Cdk intermittently, allowing re-licensing)
d) Gene amplification - successive DNA replication cycles of specific gene loci (needed for high
rates of protein synthesis). Example: Drosophila chorion gene loci. Probably involves local
modification of chromatin to allow re-licensing. (show slides)
e) Rapid embryonic cell cycles - short S phase length, made possible by ~10X increase in the
numbers of replication origins that can be licensed and fired.
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