Cell cycle - Fudan Universityjpkc.fudan.edu.cn/picture/article/104/b3/02/864520d34f8...Discovery of...
Transcript of Cell cycle - Fudan Universityjpkc.fudan.edu.cn/picture/article/104/b3/02/864520d34f8...Discovery of...
Outline
A. Historical background
B. Phases of cell cycle
C. DNA replication
D. Telomere & telomerase
E. DNA repair
F. Mitosis & Meiosis
G. Cyclins and CDKs
H. Cell-cycle checkpoints
A. Historical background
Discovery of the cell cycle
Alma Howard & Stephen Pelc, 1953
Leland H. Hartwell, Paul M. Nurse & R. Timothy Hunt, 2001, Noble Prize
Definition of the cell cycle
• Cell cycle – from the end of division (parental cell) to the next end of division (daughter cells).
B. Phases of cell cycleC. DNA replicationD. Telomere & telomeraseE. DNA repairF. Mitosis & Meiosis
Interphase
• Interphase is the period between each mitotic cell division.
• 95% of cell cycle is interphase.
• Cell metabolism, DNA replication, RNA transcription, protein translation take place in interphase.
Interphase
• Gap 1 & Gap 2 phase (G1 & G2), cells grow & metabolize, RNA & proteins synthesis.
• Synthesis phase (S), DNA replication & repair.
G1 Phase
• Cell prepares to enter S phase.
• Time courses are cell diverse.
• Different cell types:
Cycling cell
Quiescent cell (G0 cell)
Terminal differentiation cells
Restriction point
• Restriction point (eukaryote cells), Check point (yeast)
S Phase
• DNA replication
• Centrosome replication
• Histone synthesis
• Nucleosome package
C. DNA replication
DNA replication
• DNA replication takes place in S (synthesis) phase of interphase.
Start of DNA replication
• Origin• Replication fork• Replication bubbleStart in bi-directionStart at different
time
DNA polymerase
Type DNA Polymerase α
DNA Polymerase β
DNA Polymerase γ
DNA Polymerase δ
DNA Polymerase ε
Location nucleus nucleus mitochondria nucleus nucleus
Function replication,bind primase
(synthesis RNA pimer)
DNA repair replication & maintaining
mitochondria genome
elongation Fill gap, recombination,
repair
5’ to 3’ polymerase
+ + + + +
3’ to 5’ exon exonuclease
- - + + +
5’ to 3’ exon exonuclease
- - - - -
Priming & Replisome
• RNA primer, primase, primosome
• Replisome 5’ to 3’ replication Semi-conservative
replication
Extend of DNA strand
Extend of DNA strand
• Semi-discontinuous extension
• leading strand & lagging strand
• Okazaki fragment
Stop of DNA replication
• Two opposite direction replication forks meet or the replication fork meets a “stop DNA replication sequence”.
• Nucleosome of parent chromatin open one by one.
• Parent histone move to daughter leading strand. Lagging strand new histone is synthesis.
• Histone octamer don’t separate, total conservative replication
D. Telomere & telomerase
Elizabeth H. Blackburn Carol W. Greider Jack W. Szostak 2009
Telomere & telomerase
Clinical implications
• Aging: organ regeneration therapies, progeria, extend lifespan
• Cancer
• Heart disease, diabetes, psychological stress
Special features of replication
• Bi-directional initiation
• Semi-conservative replication
• Semi-discontinuous extension
E. DNA repair
DNA repair
• Photo reactivation
• Excision repair
• Recombination repair
• Induction repair
Dark repair
Clinical implications
• Skin cancer: excision repair deficient
• Breast cancer : recombination repair deficient, BRCA-2
G2 Phase
• DNA copies duplicated from 2n to 4n.
• Cell growth continues.
• Enzymes and other proteins are synthesized for cell division
F. Mitosis & Meiosis
M Phase
• Cytoplasm division (cytokinesis)
• Nuclear division (karyokinesis)
Identical genotypes but different phenotypes in daughter cells, eg: Stem cells
Prophase
• Chromatin coils.
• Centromere & kinetochore appears
• Nucleus disappears.
• Microtubule forms
• Centrosome migrate.
Prophase is the longest phase of mitosis.
Prometaphase
• Nuclear membrane and lamina break down
• X shape chromosome forms
• Microtubule capture chromosome
• Kinetochore microtubule & polar microtubule form
• Spindle forms
Breakdown and re-formation
of Nuclear lamina
Metaphase
• Chromatids attach to spindle fibers.
• Chromatids alignment to equatorial plate of spindle.
Anaphase
• Centromere splits.
• Chromatids separate to chromosomes.
Telophase
• Nuclear membrane and lamina reform.
• Chromosomes uncoil.
• Kinetochore microtubule disappear, Polar microtubule elongate
Cytokinesis
• Equatorial plate constricts to form furrow.
• Actin & myosin filaments forms contractile ring.
Meiosis
• Meiosis is a special form of mitosis in eukaryotes cells.
• One DNA replication, twice division.
• Special features: homologue chromosomes pair, synapsis, recombination.
• Evolutional role: Reduce DNA from 4N to 1N provide genetic stability, recombination provide genetic diversity.
Comparison between mitosis & Meiosis
Stages of meiosis
• Premeiotic interphase: G1, S, G2
• Meiosis I: prophase, prometaphase, metaphase, anaphase, telophase, cytokinesis
• Meiosis I Prophase: leptotene phase, zygotene phase, pachytene phase, diplotene phase, diakinesis phase
• Interkinesis
• Meiosis II
Key stages of meiosis
Meiosis I Prophase
• Leptotene phase: chromatins condense (two chromatids stick together)
• Zygotene phase: homologous chromosomes pair, bivalent, synapsis, synaptonemal complex, (DNA replicate)
• Pachytene phase: recombination, histone synthesis
• Diplotene phase: homologous chromosome separate, chiasma.
• Diakinesis phase: chiasma terminalization
Other stages in meiosis I
• Meiosis I metaphase: tetrad, 4 kinetochore
• Meiosis I anaphase: random
8.4 million combination + recombination + random mating of sperm & oocyte = uniquegamete
• Interkinesis / no interkinesis.
Meiosis II
• Meiosis II: spermatogenesis, oogenesis
• Spermatogenesis: spermatoponium, primary & secondary spermatocyte, spermatid, sperm
• Oogenesis: oogonium, primary & secondary oocyte, meiotic arrest at prophase I, polar body
Oogenesis & spermatogenesis
Overview of mitosis & meiosis
G. Cyclins and CDKs
Cell cycle regulation discovery
• Hartwell, 1960s
• Identified CDC (cell division cycle ) genes.
• Identified Cdc28, codes p34/cdc28 protein, start gene, G1→S
• Identified checkpoints
Cell cycle regulation discovery
• Nurse, 1970s
• Identified cdc2, G2→M & G1→S.
• Isolated the first cdc gene, cdc2, codes protein p34/cdc2
• Isolated the first human homolog gene, coding CDK1 protein
• CDK activation is dependent on phosphorylation.
Cell cycle regulation discovery
• Hunt, 1980s
• Identified cyclin genes
• The concentration of cyclins rise and fall in a
predictable pattern as the cell cycles progress.
Mitosis promoter factor (MPF)
• Johnson & Rao, Masui & Markert, 1970s
• MPF = p32 + p45
• MPF = Cdc2 + cyclin B
Cyclin
• Mammalian cyclin A, B, C, D, E, F, G, H
• Cyclins are synthesized at specific stages of the cell cycle.
CDK
• CDK (cyclin-dependent kinases, human homolog protein), CDC (cell division cycle gene, yeast genes)
• Mammalian CDK1, 2, 3, 4, 5, 6, 7, 8
• CDK is serine / threonine kinase.
• CDKs are constitutively expressed.
Cyclin and CDK
• Cyclin is the regulatory subunit of the cyclin / CDK complex, CDK is the catalytic subunit
• They form heterodimer complex through Cyclin box and CDK kinase domain.
CDK activity is dependent upon Cyclin
• The cyclins accumulate throughout interphase and are rapidly degraded toward the end of mitosis.
• CDK Kinase activity reaches maximum when bind to cyclin.
Cyclin and CDK
ComplexVertebrate Yeast
Cyclin CDK Cyclin CDK
G1-CDK Cyclin D CDK4 、6 Cln 3 CDK1(CDC28)
G1/S-CDK Cyclin E CDK2 Cln 1、2 CDK1(CDC28)
S-CDK Cyclin A CDK2 Clb 5、6 CDK1(CDC28)
M-CDK Cyclin B CDK1(CDC2) Clb 1-4 CDK1(CDC28)
Different Cyclin / CDK complex
G1->S
• Go through G1 restriction point is controlled by complexes of Cdk4 and Cdk6 with cyclin D.
• Cdk2/cyclin E complexes function in late G1 and are required for the G1 to S transition.
G2->M
• Cdk2/cyclin A complexes are then required for progression through S phase.
• CDK1/cyclin B complexes drive the G2 to M transition.
CDK activation
• Step 1. CDK1 forms complexes with cyclin B during S and G2 phases, no kinase activity.
CDK activation
• Step 2. Weel/mik1 kinase, CDK activting kinase phosphorylate CDK1 on threonine-161, as well as on tyrosine-15 (and threonine-14 in vertebrate cells), no kinase activity.
• Step 3. Cdc25c dephosphorylation of Thr14 and Tyr15 activates MPF at the G2 to M transition.
CDK activation
CDK activation
• Regulation of MPF
CDK activator
• Weel1 helps the phosphorylation of Thr14 and Tyr15 while Cdc25 phosphatase helps the dephosphorylation
CDK inhibitors• Cyclin-dependent kinase inhibitors, CDKI
• The CIP/KIP family includes the genes p21, p27 and p57, inhibit CDK2, CDK3 , CDK4, CDK6
• The INK4a family includes p16, p15, p18, p19 , inhibit CDK4, CDK6
Targets of CDK
• Activate other protein kinases.
• Phosphorylate structural proteins.
H. Cell-cycle checkpoints
Cell-cycle checkpoints
• G1-S checkpoint: Restriction point / start
• S checkpoint
• G2-M checkpoint
• M checkpoint: Spindle checkpoint
DNA damage checkpoints
• Sensors of damage: ATM, ATR
• Signal transducers: CHEK1, CHEK2
• Effectors: p53, cdc25, pRb
• Mediators: BRCA1, Clapin, 53BP1, MDC1
ATM & ATR
• ATM (ataxia-telangiectasa mutated)
• ATR (ATM and Rad3 related)
p53
pRb
• Retinoblastoma, “two-hit” theory of cancer, 1970s
Clinical implication
• Leukemia: ataxia-telangiectasa
• Cancer
Roles of cell cycle regulation
• Active / inactive proteins in specific phase of cell cycle in an ordered and directional way (positive control)
• Prevent uncontrolled cell division, block cell cycle at checkpoints in specific phase to detect and repair DNA damage (negative control)
• Response to the external stimulus or stress (response)
• Cell cycle regulation
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