Cell cycle. Chen Li. Department of cellular and genetic medicine

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1 Cell cycle Chen Li Department of cellular and genetic medicine

2 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

3 A. Historical background

4 Discovery of the cell cycle Alma Howard & Stephen Pelc, 1953 Leland H. Hartwell, Paul M. Nurse & R. Timothy Hunt, 2001, Noble Prize

5 Definition of the cell cycle Cell cycle from the end of division (parental cell) to the next end of division (daughter cells).

6 B. Phases of cell cycle C. DNA replication D. Telomere & telomerase E. DNA repair F. Mitosis & Meiosis

7 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.

8 Interphase Gap 1 & Gap 2 phase (G1 & G2), cells grow & metabolize, RNA & proteins synthesis. Synthesis phase (S), DNA replication & repair.

9 Cell prepares to enter S phase. Time courses are cell diverse. Different cell types: Cycling cell Quiescent cell (G0 cell) Terminal differentiation cells G1 Phase

10 Restriction point Restriction point (eukaryote cells), Check point (yeast)

11 S Phase DNA replication Centrosome replication Histone synthesis Nucleosome package

12 C. DNA replication

13 DNA replication DNA replication takes place in S (synthesis) phase of interphase.

14 Start of DNA replication Origin Replication fork Replication bubble Start in bi-direction Start at different time

15 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 5 to 3 polymerase 3 to 5 exon exonuclease 5 to 3 exon exonuclease replication, bind primase (synthesis RNA pimer) DNA repair replication & maintaining mitochondria genome elongation Fill gap, recombination, repair

16 RNA primer, primase, primosome Replisome 5 to 3 replication Semi-conservative replication Priming & Replisome

17 Extend of DNA strand

18 Extend of DNA strand Semi-discontinuous extension leading strand & lagging strand Okazaki fragment

19 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

20 D. Telomere & telomerase

21 Telomere & telomerase Elizabeth H. Blackburn Carol W. Greider Jack W. Szostak 2009

22 Clinical implications Aging: organ regeneration therapies, progeria, extend lifespan Cancer Heart disease, diabetes, psychological stress

23 Replication of telomeres

24 Special features of replication Bi-directional initiation Semi-conservative replication Semi-discontinuous extension

25 E. DNA repair

26 DNA repair Photo reactivation Excision repair Recombination repair Induction repair Dark repair

27 Clinical implications Skin cancer: excision repair deficient Breast cancer : recombination repair deficient, BRCA-2

28 G2 Phase DNA copies duplicated from 2n to 4n. Cell growth continues. Enzymes and other proteins are synthesized for cell division

29 F. Mitosis & Meiosis

30 M Phase Cytoplasm division (cytokinesis) Nuclear division (karyokinesis) Identical genotypes but different phenotypes in daughter cells, eg: Stem cells

31 Chromatin coils. Centromere & kinetochore appears Nucleus disappears. Microtubule forms Centrosome migrate. Prophase is the longest phase of mitosis. Prophase

32 Prometaphase Nuclear membrane and lamina break down X shape chromosome forms Microtubule capture chromosome Kinetochore microtubule & polar microtubule form Spindle forms

33 Breakdown and re-formation of Nuclear lamina

34 Metaphase Chromatids attach to spindle fibers. Chromatids alignment to equatorial plate of spindle.

35 Anaphase Centromere splits. Chromatids separate to chromosomes.

36 Telophase Nuclear membrane and lamina reform. Chromosomes uncoil. Kinetochore microtubule disappear, Polar microtubule elongate

37 Cytokinesis Equatorial plate constricts to form furrow. Actin & myosin filaments forms contractile ring.

38 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.

39 Comparison between mitosis & Meiosis

40 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

41 Key stages of meiosis

42 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

43 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 = unique gamete Interkinesis / no interkinesis.

44 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

45 Oogenesis & spermatogenesis Overview of mitosis & meiosis

46 G. Cyclins and CDKs

47 Cell cycle regulation discovery Hartwell, 1960s Identified CDC (cell division cycle ) genes. Identified Cdc28, codes p34/cdc28 protein, start gene, G1 S Identified checkpoints

48 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.

49 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.

50 Mitosis promoter factor (MPF) Johnson & Rao, Masui & Markert, 1970s MPF = p32 + p45 MPF = Cdc2 + cyclin B

51 Cyclin Mammalian cyclin A, B, C, D, E, F, G, H Cyclins are synthesized at specific stages of the cell cycle.

52 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.

53 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.

54 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.

55 Cyclin and CDK

56 Different Cyclin / CDK complex Vertebrate Yeast Complex Cyclin CDK Cyclin CDK G 1 -CDK Cyclin D CDK4 6 Cln 3 CDK1(CDC28) G 1 /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)

57 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.

58 G2->M Cdk2/cyclin A complexes are then required for progression through S phase. CDK1/cyclin B complexes drive the G2 to M transition.

59 CDK activation Step 1. CDK1 forms complexes with cyclin B during S and G2 phases, no kinase activity.

60 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.

61 CDK activation Step 3. Cdc25c dephosphorylation of Thr14 and Tyr15 activates MPF at the G2 to M transition.

62 Regulation of MPF CDK activation

63 CDK activator Weel1 helps the phosphorylation of Thr14 and Tyr15 while Cdc25 phosphatase helps the dephosphorylation

64 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

65 Targets of CDK Activate other protein kinases. Phosphorylate structural proteins.

66 H. Cell-cycle checkpoints

67 Cell-cycle checkpoints G1-S checkpoint: Restriction point / start S checkpoint G2-M checkpoint M checkpoint: Spindle checkpoint

68 DNA damage checkpoints Sensors of damage: ATM, ATR Signal transducers: CHEK1, CHEK2 Effectors: p53, cdc25, prb Mediators: BRCA1, Clapin, 53BP1, MDC1

69 ATM & ATR ATM (ataxiatelangiectasa mutated) ATR (ATM and Rad3 related)

70 p53

71 prb Retinoblastoma, two-hit theory of cancer, 1970s

72 Clinical implication Leukemia: ataxia-telangiectasa Cancer

73 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

74 Thanks!

Chapter 3. DNA Replication & The Cell Cycle

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