Cell cycle

Chen Li

Department of cellular and genetic medicine13 – 223 chenli2008@fudan.edu.cn

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

Replication of telomeres

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

Thanks!