Control of DNA

1. Chapter 12 & 18 Control of DNA

2. The Cell Cycle • cell cycle = ordered series of events that lead to cell division and the production of 2 daughter cells each with the same # and type of chromosomes as the parent • 2 key events in the cell cycle • 1. chromosome replication • 2. chromosome segregation • consists of two phases • Mitotic (M) phase = mitosis and cytokinesis • Interphase = cell growth and copying of chromosomes in preparation for cell division • Interphase - about 90% of the cell cycle • can be divided into sub-phases • G1 phase -“first gap” • S phase “synthesis” • G2 phase - “second gap”

3. Nobel Prize? You bet! • Leland Hartwell • Tim Hunt • Paul Nurse • 2001 https://plus.google.com/+NobelPrize/posts

4. Eukaryotic Cell Cycle Control • 2 major events take place during the cell cycle • duplication of the chromosomes (S phase) • duplicated chromosomes are distributed to the daughter cells (M phase)

5. Some terms to know -parent cell = cell about to undergo division -daughter cell = cell that results from either mitosis or meiosis -somatic cell = any cell within the body other than an egg or sperm -somatic cell has two complete sets of chromosomes -one set is called the haploid number of chromosomes (n) -therefore the cell is said to be diploid(2n) e.g. humans n = 23 (2n = 46) -germ cell or gamete = sex cell -gamete has only one set of chromosomes and is haploid

6. Eukaryotic Cell Division • two kinds of eukaryotic cell division • Mitosis • Meiosis • eukaryotic cell division consists of • Mitosis - the division of the genetic material in the nucleus • Cytokinesis - the division of the cytoplasm • mitosis described by the German anatomist Walther Flemming in 1882 • thought the cell was simply growing larger between each period of cell division • now known that the growing cells were in the G1 phase • never observed the M phase

7. Some terms to know -parent cell = cell about to undergo division -daughter cell = cell that results from either mitosis or meiosis -somatic cell = any cell within the body other than an egg or sperm -somatic cell has two complete sets of chromosomes -one set is called the haploid number of chromosomes (n) -therefore the cell is said to be diploid(2n) e.g. humans n = 23 (2n = 46) -germ cell or gamete = sex cell -gamete has only one set of chromosomes and is haploid

8. Most cell division results in genetically identical daughter cells • most cell division results in daughter cells with identical genetic information (i.e. amount and type of DNA) • the genetic information has to be duplicated and distributed amongst the two daughter cells • once the DNA is duplicated and distributed then the cell can divide • SO: cell division is not just the pinching of the parent cell into two daughter cells

9. Cellular Organization of the Genetic Material • all the DNA in a cell constitutes the cell’s genome • REMINDER: when not dividing – much of the eukaryotic DNA is in its loosest formation = chromatin • a complex of DNA and protein that condenses during cell division • allows access to the machinery for DNA replication and transcription 20 m

10. in preparation for cell division - DNA is replicated and condenses into chromosomes • chromosome = organized structure of DNA and protein • chroma = color • soma = body • the building material of a chromosome is chromatin • each duplicated chromosome is made of two sister chromatids = joined copies of the original chromosome • these chromatids will separate during cell division and be partitioned into each daughter cell • chromatids are joined by a structure called a centromere Sisterchromatids Centromere 0.5 m every eukaryotic species has a characteristic number of chromosomes in each cell nucleus e.g. humans – n=23 e.g. drosophila – n=2 e.g. dog – n=39

11. Centromere • location along the chromosome – two sister chromatids are joined • condensed region within the chromosome • responsible for the accurate segregation of sister chromatids during mitosis & meiosis • shared by sister chromatids during mitosis • site of the centromere where spindle microtubules attach – area of DNA and protein = kinetochore Sisterchromatids Centromere 0.5 m

12. Outer Plate Microtubules Inner Plate • Kinetochore • a structure of DNA (CEN DNA) and proteins located in the centromere • for the attachment of the chromosome to the spindle during mitosis and meiosis • one MT attaches to one kinetochore on one chromatid • a 2nd MT attaches to the kinetochore on the other chromatid • attachment of these MTs results in movement toward the poles • a “tug of war” results – chromosomes move back and forth and eventually settle in the metaphase plate Chromatid Microtubules Kinetochore

13. Chromosome and Chromosome: Confusion!!! • prior to cell division – the duplicated chromatin condenses into its most dense form = chromosome • two sister chromatids joined by a centromere • typically called a duplicated chromosome • during cell division - the two sister chromatids separate • once separated - the chromatids are still called chromosomes

14. chromosome scaffold Control of DNA Structure: Condensation of Chromatin • shortest human chromosome is 44 million nucleotides long • this means packing 14000 um (14mm) of linear DNA into a nucleus around 2um • that is a 7000:1 packing ratio!!! • packing is achieved by the chromatin http://www.ndsu.edu/pubweb/~mcclean/plsc431/eukarychrom/eukaryo3.htm

15. chromosome scaffold Control of DNA Structure: Condensation of Chromatin • nucleosome = DNA helix wrapped around a histone protein core • responsible for organizing the DNA as chromatin • 10 nm and 30 nm fibers • other proteins in nuclear lamina are involved organizing chromatin as the chromosome • these nuclear lamina proteins = non-histone proteins • NHPs form a chromosome scaffold • the heterochromatin is “looped” onto this scaffold • megabase, long-loops of 30nm heterochromatin associated with this scaffold  300nm fiber

16. chromosome scaffold Control of DNA Structure: Condensation of Chromatin • specific sequences in the chromosome called scaffold-associated regions or SARs interact with the scaffold to create the 300 nm fiber • last level of “packing” is a 700 nm fiber • not much known about this • the chromosome seen in metaphase

17. Mitosis is conventionally divided into five phases • Prophase • Prometaphase • Metaphase • Anaphase • Telophase • Cytokinesis overlaps the latter stages of mitosis

18. 10 m G2 of Interphase Prophase Prometaphase Metaphase Anaphase Telophase and Cytokinesis Centrosomes(with centriole pairs) Chromatin(duplicated) Fragments of nuclearenvelope Nonkinetochoremicrotubules Early mitoticspindle Aster Metaphase plate Cleavagefurrow Nucleolusforming Centromere Plasmamembrane Nuclearenvelope Chromosome, consistingof two sister chromatids Kinetochore Kinetochoremicrotubule Nucleolus Nuclearenvelopeforming Daughterchromosomes Centrosome atone spindle pole Spindle

19. Mitosis http://www.loci.wisc.edu/outreach/bioclips/CDBio.html • Prophase: prior to prophase, the replicated DNA is starting to condense into sister chromatids joined at the centromere  (duplicated) chromosome 1. the centrioles (replicated at G2) move apart from each other 2. the nucleoli disappear Spindle – structure that includes the two centrioles, two asters and the spindle microtubules than span the cell Aster – a radial array of short MTs extending from the centrioles

20. Spindle Formation 3. the kinetochore forms in the centromere 4. the spindle forms between the centrioles (made of microtubules) -the centrioles are not essential for spindle formation; plant cells do not have centrioles -spindle MT assembly results from the polymerization of tubulin subunits -other MTs of the cytoskeleton disassemble to provide more tubulin to the spindle Spindle – structure that includes the two centrioles, two asters and the spindle microtubules that span the cell Aster – a radial array of short MTs extending from the centrioles

21. Metaphase: centrioles are at opposite ends of the cell and the spindle is complete 1. the chromosomes move and line up along a central zone= metaphase plate -the tug of war at pro-metaphase eventually positions the chromosomes midway alone the length of the cell 2. non-kinetochore MTs interact with the opposite pole & the aster MTs make contact with the plasma membrane – the spindle is now complete 10 m 3 Metaphase

22. 4. Anaphase: shortest of the mitotic phases 1. the chromatid pairs separate into daughter chromosomes 2. one chromatid/chromosome moves toward one centriole of the cell, the other the opposite -pulled apart by the action of the spindle – the kinetochore MTs begin to shorten -PLUS non-kinetochore MTs grow – this elongates the cell ** At the end of this phase – each end of the cell has equivalent numbers of chromosomes – same number as the parent cell **the sister chromatids separate because of enzymatic activity -an enzyme called separase cleaves a protein known as cohesin (protein in the centromere that holds the sister chromatids together) -separates the sister chromatids

23. 4. Telophase:reverse of Prophase 1. nuclear envelope reforms – two daughter nuclei result -part of the new nuclear membrane is recycled from the old fragments, other parts are made new by the cell 2. the nucleoli reappear 3. the spindle disappears as the MTs depolymerize 4. daughter chromosomes uncoil ** Cytokinesis starts during late anaphase and is well underway during telophase

24. (a) Cleavage of an animal cell (SEM) Cytokinesis:division of cytoplasm -separates the parent into two daughter cells -differs in animal cells and plant cells Animal cell Cytokinesis: results from cleavage -pinches into two daughters -actin filaments assemble to form a contractile ring along the equator of the cell -actin interacts with myosin proteins – causes the ring to contract -actin-myosin interaction first forms a “cleavage furrow”- slight indentation around the circumference of the cell -continued interaction divides the cell by a “purse string” mechanism 100 m Cleavage furrow Daughter cells Contractile ring ofmicrofilaments

25. Plant cell Cytokinesis: No cleavage furrow possible -vesicles bud from the Golgi apparatus and migrate to the middle of the cell -vesicles coalesce to produce a cell plate -other vesicles fuse to the plate bringing in new building materials -cell plate grows and eventually splits the cell into two daughter cells (b) Cell plate formation in a plant cell (TEM) 10 m Cell plate Vesiclesformingcell plate Wall of parent cell 1 m New cell wall Cell plate 5 Telophase Daughter cells

28. Cell wall Origin ofreplication Plasma membrane Binary Fission in Bacteria E. coli cell Bacterial chromosome 1 Chromosomereplicationbegins. Two copies of origin • bacteria and archaea reproduce by binary fission • the chromosome replicates and the two daughter chromosomes actively move apart • the plasma membrane pinches inward, dividing the cell into two • BUT there is no ordered segregation of the duplicated chromosome • duplicate it and divide it into two cells 2 Origin Origin Replicationcontinues. 3 Replicationfinishes. 4 Two daughtercells result.

29. Diatoms andsome yeasts (c) Bacterialchromosome (a) Bacteria The Evolution of Mitosis Chromosomes Microtubules (b) Dinoflagellates • mitosis probably evolved from binary fission • certain protists exhibit types of cell division that seem intermediate between binary fission and mitosis Intact nuclearenvelope Kinetochoremicrotubule Intact nuclearenvelope Kinetochoremicrotubule (d) Most eukaryotes Fragments ofnuclear envelope

30. Phases of the Cell Cycle • consists of two phases • Mitotic (M) phase = mitosis and cytokinesis) • Interphase = cell growth and copying of chromosomes in preparation for cell division • Interphase - about 90% of the cell cycle • can be divided into sub-phases • G1 phase -“first gap” • S phase “synthesis” • G2 phase - “second gap”

31. Phases of the Cell Cycle • G1 phase - time in phase depends on species • normal cell functions & growth in size • mRNA and protein synthesis in preparation for S phase • critical phase in which cell commits to division or leaves the cell cycle to enter into a dormancy phase (G0) • S phase - 6 to 8 hours • synthesis of histone proteins & DNA replication • G2 phase – 2 to 5 hours • rapid cell growth • protein synthesis in preparation for M phase • duplication of the centrioles/centrosomes http://www.wisc-online.com/objects/index.asp?objID=AP13604

32. Cell Cycle Checkpoints • interphase not only allows the cell to perform its normal functions but also allows the cell to check whether it is ready to enter mitosis • cell cycle is controlled by a control system that coordinates and triggers key events in the cell cycle • progression through the cell cycle requires a combination of internal and external signals • these signals control whether the cell is ready to continue on into the S and M phases

33. EXPERIMENT Experiment 1 Experiment 2 Eukaryotic Cell Cycle Control M G1 S G1 • EXPERIMENT: fusion of two cells at different points in the cell cycle • Results in “re-setting” of one cell to coordinate with the other • CONCLUSION: cell division is driven by specific chemical signals present in the cytoplasm RESULTS S S M M When a cell in the Sphase was fusedwith a cell in G1,the G1 nucleusimmediately enteredthe S phase—DNAwas synthesized. When a cell in the M phase was fused witha cell in G1, the G1nucleus immediatelybegan mitosis—a spindleformed and chromatincondensed, even thoughthe chromosome had notbeen duplicated.

34. Cell Cycle Checkpoints • so the control system of the cell cycle monitors these signals and determines whether to proceed through the cell cycle • there are specific points along the cell cycle where “decisions” are made by this control system = CHECKPOINTS • cancer cells manage to escape the usual controls on the cell cycle

35. G1 checkpoint Cell Cycle Checkpoints • major checkpoints – G1, G2 and M • G1 checkpoint – G1/S progression through a point called the restriction point or START point of the cell cycle • G2 checkpoint – G2/M progression which will lead to the start of mitosis and chromosome alignment • M checkpoint – Metaphase to Anaphase transition where chromatid separation occurs Controlsystem S G1 G2 M M checkpoint G2 checkpoint

36. Cell Cycle Checkpoints G0 • for many cells, the G1/S checkpoint seems to be the most important • if a cell receives a go-ahead signal at this G1/S checkpoint  will usually complete the S, G2, and M phases and divide • many texts call this checkpoint the Start (yeasts) or Restriction point (mammalian cells) • if the cell does not receive the go-ahead signal - will exit the cycle, switching into a non-dividing state called the G0 phase • most cells in the body are in the G0 phase and remain there • some cells have the ability to leave G0 and re-enter the cell cycle G1 checkpoint G1 G1 (a) Cell receives a go-ahead signal. (b) Cell does not receive a go-ahead signal.

37. IN ANIMAL CELLS A “GO-AHEAD” SIGNAL MUST BE PRODUCED TO OVERRIDE A BUILT IN “STOP” SIGNAL

38. Cell Cycle Control System • the proteins of this system evolved over a billion years ago • so well conserved in eukaryotes – take from human control cells and put into yeast cells – they work!! • much of the early research done –done in yeast • search for mutations in genes that encode critical parts of the cell cycle control system = cell-division-cycle genes or cdc genes • many mutations in this family of genes causes the cell cycle to arrest at specific points – such as checkpoints • additional work done in frog eggs and in mammalian cell cultures • e.g. immortalized mammalian cell lines

39. The Cell Cycle Control System: Cyclins and Cyclin-Dependent Kinases • two types of regulatory proteins are involved in cell cycle control: cyclins and cyclin-dependent kinases (Cdks) • cyclins named for the cyclical changes in their concentration through the cell cycle • cdks named because their phosphorylation activities requiring their binding to their “partner” cyclin • cdks cannot work as kinases unless they are bound to their partner cyclin the cdk-cyclin complex is called a heterodimer

40. Cyclins and Cyclin-Dependent Kinases • cyclin/CDKs regulate the activities of multiple proteins by the cdk phosphorylating them • Cyclin/CDK partners are involved in: • 1. entry to the cell cycle (i.e. G1 phase) • 2. DNA replication (i.e. S phase) • 3. segregation of chromosomes during mitosis (i.e. M phase)

41. The Cell Cycle Control System: Cyclins and Cyclin-Dependent Kinases • cdk activity fluctuates during the cell cycle • cdk proteins are expressed first • activity is controlled by an array of proteins - including the cyclins • cyclical changes in cdk activities leads to cyclical changes in the phosphorylation of their target proteins • cdks phosphorylate key proteins responsible for passing through each checkpoint

42. The Cell Cycle Control System: Cyclins and Cyclin-Dependent Kinases • eukaryotic cells have four classes of cyclins • each act at a specific stage of the cell cycle • eukaryotic cells require three of these classes for their cell cycle • 1. G1/S cyclins • 2. S-cyclins • 3. M-cyclins

43. The Cell Cycle Control System: Cyclins and Cyclin-Dependent Kinases • 1. G1/S cyclins – active in late G1; trigger the progression through the G1 restriction point (START) • 2. S-cyclins – activate cdks that help stimulation chromosome duplication + they help initiate mitosis • e.g. the cdk phosphorylates proteins that activate DNA helicases G1/S cyclins: levels peak during G1 S cyclins: levels slowly climb and stay high until Mitosis

44. The Cell Cycle Control System: Cyclins and Cyclin-Dependent Kinases • 3. M-cyclins – activate the cdks that stimulate progression through the G2 checkpoint and into Mitosis • Cdk phosphorylate numerous proteins involved in chromosome segregation and other aspects of mitosis M cyclins: levels climb during G2 and drop just after metaphase checkpoint

45. Cyclins and Cyclin-Dependent Kinases – no you don’t have to know these for your exam!!! FUNCTION G1/entry into cell cycle Entry into cell cycle & S phase Mitosis modified from Cell Biology Alberts et al.

46. Cyclins and CDKs in action • work in frog eggs identified a cdk-cyclin complex that triggered the cell’s passage past the G2 checkpoint into the M phase • called MPF (maturation-promoting factor) • also called Mitosis-promoting factor • cyclin B and CDK1 • now known to act in all eukaryotic mitotic cells G2 G2 G1 G1 M S G1 S M M MPF activity Cyclinconcentration Time (a) Fluctuation of MPF activity and cyclin concentration during the cell cycle

47. Cyclins and CDKs in action • cyclin B synthesis begins in late S phase and increases through G2 and into M • cyclin B combines with CDK1 in G2 to produce MPF • when enough MPF is made – the cell passes the G2 checkpoint and enters Mitosis • MPF promotes Mitosis via CDK1 phosphorylating its target proteins • e.g. phosphorylated target proteins results in nuclear envelope fragmentation

48. G2 G2 G1 G1 M S G1 S M M MPF activity Regulation of CDK activity Cyclinconcentration • want to control the CDK? control the expression of the cyclin!!! • the most important regulatory control of a cyclin is its degradation • e.g. during anaphase cyclin B becomes degraded and CDK1 activity starts to fall • the cdk1 part of the MPF actually degrades its partner cyclin • the cdk1 is “recycled” for future cycles • M phase stops and G1 begins Time (a) Fluctuation of MPF activity and cyclin concentration during the cell cycle G1 S Cdk Cyclin accumulation M G2 Degradedcyclin G2checkpoint Cdk Cyclin isdegraded Cyclin MPF (b) Molecular mechanisms that help regulate the cell cycle

49. Degradation of cyclins and cell cycle control • activation of specific cyclin-cdk complexes also drive progression through the G1 and G2 checkpoints • progression through the M checkpoint requires the degradation of proteins • e.g. degradation of cyclin B • one key regulator of cyclin degradation is the anaphase-promoting complex or APC • mitotic cdk/cyclin complexes induce chromosome condensing, breakdown of the nuclear envelope, assembly of the spindle and the alignment of chromosomes • this gets you up to Anaphase • once this happens – the sister chromatids must separate at Anaphase • the APC controls this

50. Degradation of cyclins and cell cycle control cessation of mitosis • mitotic cdk/cyclins activate the APC (via phosphorylation) • the APC directs the degradation of proteins that act as anaphase inhibitors in the cell • allows the onset of anaphase cyclin/cdk activates APC degradation of inhibitors anaphase