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The Cell Cycle, DNA Replication, and Mitosis

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1 The Cell Cycle, DNA Replication, and Mitosis
Chapter 19 The Cell Cycle, DNA Replication, and Mitosis

2 The Cell Cycle, DNA Replication, and Mitosis
Cell growth is generally accompanied by cell division, whereby one cell gives rise to two new daughter cells All the genetic information in the nucleus must be accurately duplicated and carefully distributed to the daughter cells In doing this a cell passes through a series of stages known as the cell cycle 2

3 Overview of the Cell Cycle
The cell cycle begins when two new cells are formed by division of a parent cell and ends when one of these cells divides again M phase is when the cells actually divide; the nucleus first, followed by the cytoplasm Nuclear division is mitosis and division of the cytoplasm is cytokinesis 3

4 Chromosomes in mitosis
At the beginning of mitosis, chromatin folds and condenses to produce visible chromosomes DNA has replicated, so each chromosome is composed of two sister chromatids The microtubules of the mitotic spindle will distribute the chromatids to opposite ends of the cell

5 Figure 19-1A

6 Mitosis is a relatively short part of the cell cycle
Cells spend very little time in M phase Most of the time is spent in interphase, which is composed of G1 phase, S phase (when DNA is replicated), and G2 phase The overall length of the cell cycle is called the generation time; in cultured mammalian cells this is about 18–24 hours

7 Figure 19-1B

8 The G phases G1 is quite variable depending on cell type; G2 is shorter and less variable During G1 a major decision is made, whether a cell will divide again; cells that arrest in G1, waiting for a signal to divide, are said to be in G0 Cells that exit the cell cycle are said to undergo terminal differentiation

9 DNA Replication DNA replication is a central event in the cell cycle
The underlying mechanism depends on the double-helical structure of DNA One strand of every new DNA molecule is derived from the parent molecule and the other is new: semiconservative replication

10 Figure 19-2 10

11 DNA Replication Is Usually Bidirectional
DNA replication is especially well understood in Escherichia coli Saccharomyces cerevisiae and the virus SV40 are used in studies of eukaryotic replication Replication is very similar in prokaryotes and eukaryotes

12 Early experiments in replication
Cairns studied replication in E. coli; he grew cells in a medium containing 3H-thymidine He visualized the circular chromosomes by autoradiography; he observed replication forks These are formed where replication begins and then proceeds in bidirectional fashion away from the origin

13 Figure 19-4A

14 Bacterial replication
Replication forks move away from the origin, unwind the DNA, and copy both strands as they proceed This is called theta () replication and is observed in replication of circular DNA molecules The two copies of the replicating chromosome bind to the plasma membrane at their origins; when replication is complete the cell divides by binary fission

15 Figure 19-4B

16 Figure 19-4C

17 Eukaryotic DNA Replication Involves Multiple Replicons
In eukaryotes replication of linear chromosomes is initiated at multiple sites, creating replication units called replicons At the center of each replicon is a DNA sequence called an origin of replication, where synthesis is initiated by several groups of initiator proteins First, a multisubunit protein complex called the origin recognition complex (ORC) binds the replication origin 17

18 Figure 19-5A-E

19 Figure 19-5A

20 Figure 19-5B

21 Figure 19-5C

22 Figure 19-5D

23 Figure 19-5E

24 Eukaryotic replication (continued)
Next, the minichromosome maintenance (MCM) proteins bind the origin The MCM proteins include several DNA helicases that unwind the double helix; a set of proteins called helicase loaders recruit the MCM proteins At this point all the DNA-bound proteins make up the pre-replication complex and the DNA is “licensed” for replication

25 Origins of replication
DNA sequences that act as replication origins are greatly varied in eukaryotes Sequences that confer ability to replicate when introduced into DNA molecules are called autonomously replicating sequences or ARS After replication begins, two replication forks synthesize DNA in opposite directions, forming a replication bubble that grows as replication proceeds

26 Rates of replication S phase is very rapid in embryonic cells, where cell divisions occur in quick succession, but slower in adult cells, in which fewer and more widely spaced replicons are used During S phase in eukaryotes, not all replicons are activated at the same time; some are replicated early and others later Genes that are transcriptionally active are replicated earlier than inactive genes

27 Replication Licensing Ensures That DNA Molecules Are Duplicated Only Once Prior to Each Cell Division Licensing is provided by binding of MCM proteins to the origin, which requires both ORC and helicase loaders It ensures that after DNA is replicated at each origin, the DNA cannot be licensed for replication again until after mitosis After replication begins, the MCM proteins are removed from the origins and cannot bind again

28 Role of Cdk Cyclin-dependent kinase, cdk, has many roles in the cell cycle One form produced early in S phase activates DNA synthesis at licensed origins and prevents origins from being licensed again It catalyzes phosphorylation of ORC proteins and helicase loaders

29 Geminin Multicellular eukaryotes contain an additional inhibitor of relicensing called geminin It is made during S phase that blocks the binding of MCM proteins to DNA When the cell completes mitosis, geminin is degraded and Cdk activity falls so that licensing can occur for the next cycle

30 Figure 19-6

31 DNA Polymerases Catalyze the Elongation of DNA Chains
DNA polymerase is an enzyme that can copy DNA molecules Incoming nucleotides are added to the 3 hydroxyl end of the growing DNA chain, so elongation occurs in the 5 to 3 direction Several other forms of DNA polymerase have been identified; the original is now called DNA polymerase I

32 Figure 19-7

33 Table 19-1

34 Temperature-sensitive mutations
It is difficult to grow and study mutant strains that lack important functions such as DNA replication One approach involves using temperature-sensitive mutants, which produce proteins that function properly at 37oC but lose their function when the temperature is raised to 42oC

35 Observations on temperature- sensitive bacteria
Bacterial strains have been identified in which DNA polymerase III functions normally at 37oC but loses its ability to replicate when the temperature is raised to 42oC The observations indicate that DNA polymerase III is central to bacterial DNA replication However, a variety of other proteins is needed for replication

36 Eukaryote DNA polymerases
Eukaryotic cells contain several types of DNA polymerase; more than a dozen Of these, DNA polymerase , , and  are involved in nuclear DNA replication DNA polymerase  is used in mitochondrial DNA replication Those types remaining are involved in DNA repair or replication across regions of DNA damage

37 Biotechnology functions of DNA polymerases
DNA polymerases have practical applications in biotechnology The polymerase chain reaction is a technique in which a DNA polymerase is used to amplify tiny samples of DNA

38 DNA Is Synthesized as Discontinuous Segments That Are Joined Together by DNA Ligase
DNA is synthesized in the 5 to 3 direction, but the two strands of the double helix are oriented in opposite directions One strand (the lagging strand) is synthesized in discontinuous fragments called Okazaki fragments The other (the leading strand) is synthesized in a continuous chain

39 Okazaki’s experiments
Okazaki isolated DNA from bacteria that were briefly exposed to a radioactive substrate incorporated into newly made DNA Much of the radioactivity was located in small fragments about 1000 nucleotides long With longer labeling the radioactivity became associated with longer molecules; this conversion did not take place in bacteria lacking DNA ligase

40 Figure 19-8

41 Figure 19-8A

42 Figure 19-8B

43 Okazaki’s observations illustrate how lagging strand synthesis occurs
DNA synthesis from the lagging strand is synthesized in Okazaki fragments These are then joined by DNA ligase to form a continuous new 3 to 5 DNA strand Okazaki fragments are 1000–2000 nucleotides long in bacteria and viruses, but about one-tenth this length in eukaryotic cells

44 Figure 19-9

45 Proofreading Is Performed by the 3→ 5 Exonuclease Activity of DNA Polymerase
About 1 of every 100,000 nucleotides incorporated during DNA replication is incorrect Such mistakes are usually fixed by a proofreading mechanism Almost all DNA polymerases have a 3 → 5 exonuclease activity

46 Proofreading Exonucleases degrade nucleic acids from the ends of the molecules Endonucleases make internal cuts in nucleic acid molecules The exonuclease activity of DNA polymerase allows it to remove incorrectly base-paired nucleotides and incorporate the correct base

47 Figure

48 Figure

49 RNA Primers Initiate DNA Replication
DNA polymerase can only add nucleotides to the 3 end of an existing nucleotide chain Researchers implicated RNA in the initiation process based on several observations 1. Okazaki fragments usually have short stretches of RNA at their 5 ends

50 Observations about RNA involvement in DNA replication (continued)
2. DNA polymerase is able to add nucleotides to RNA chains as well as DNA chains 3. Cells contain an enzyme called primase that synthesizes short (~10 nt) chains of RNA using DNA as a template 4. Primase is able to initiate RNA strands without a pre-existing chain to add to

51 DNA synthesis requires RNA primers
The observations led to the conclusion that DNA synthesis is initiated by the formation of short RNA primers These are synthesized by primase using a single DNA strand as the template In E. coli, primase is inactive unless accompanied by six other proteins, forming a complex called a primosome

52 Figure 19-11

53 The process of DNA synthesis
Once the RNA primer is made, DNA polymerase III (or DNA polymerase a, followed by d or e, in eukaryotes) adds deoxynucleotides to the 3 end of the primer For the leading strand, just one primer is needed, but the lagging strand needs a series of primers to initiate each Okazaki fragment When the DNA chain reaches the next Okazaki fragment the RNA is degraded and replaced with DNA; adjacent fragments are joined together by DNA ligase

54 Unwinding the DNA Double Helix Requires DNA Helicases, Topoisomerases, and Single-Stranded DNA Binding Proteins During DNA replication the two strands of the double helix must unwind at each replication fork Three classes of proteins facilitate the unwinding: DNA helicases, topoisomerases, and single-stranded DNA binding proteins DNA helicases are responsible for unwinding the DNA, using energy from ATP hydrolysis

55 Helicases The DNA double helix is unwound ahead of the replication fork, the helicases breaking the hydrogen bonds as they go In E. coli, at least two different helicases are involved; one attaches to the lagging strand and moves 5 → 3, whereas the other attaches to the leading strand and moves 3 → 5 Both are part of the primosome

56 Topoisomerases The unwinding of the helix would create too much supercoiling if not for topoisomerases These enzymes create swivel points in the DNA molecule by making and then quickly sealing double-strand or single-stranded breaks Of the ~10 topoisomerases in E. coli, the key enzyme for DNA replication is gyrase

57 Single-stranded DNA binding protein
Once strand separation has begun, molecules of SSB (single-stranded DNA binding protein) move in quickly and attach to the exposed single strands They keep the DNA unwound and accessible to the replication machinery When a segment of DNA has been replicated, the SSB molecules fall off and are recycled

58 Figure 19-12

59 Putting It All Together: DNA Replication in Summary
Starting at the origin of replication, the machinery at the replication fork adds proteins required for synthesizing DNA These are DNA helicase, DNA gyrase, SSB, primase, DNA polymerase, and DNA ligase Several other proteins are used to improve the efficiency, e.g., a ring-shaped sliding clamp keeps DNA polymerase firmly attached to DNA

60 Figure 19-13, top

61 Figure 19-13, bottom

62 The replisome Proteins involved in DNA replication are all closely associated in one large complex called a replisome The activity and movement of the replisome is powered by nucleoside triphosphate hydrolysis As the replisome moves along the DNA it must accommodate the fact that DNA is being produced on both leading and lagging strands

63 Figure 19-14

64 Eukaryotic DNA replication
Eukaryotes have much of the same replication machinery found in prokaryotes E.g. a DNA clamp protein acts along with DNA polymerase; one of these is called proliferating nuclear cell antigen (PCNA) PCNA is a clamp protein for DNA polymerase d

65 Replication factories and chromatin remodeling
Studies addressing how many origins of replication can be coordinated suggest that the immobile structures called replication factories synthesize DNA as chromatin fibers are fed through them Unfolding chromatin fibers ahead of the replication fork is facilitated by chromatin remodeling proteins that loosen nucleosome packing

66 Telomeres Solve the DNA End-Replication Problem
Linear DNA molecules have a problem in completing DNA replication on the lagging strand, because primers are required Each round of replication would end with the loss of some nucleotides from the ends of each linear molecule Eukaryotes solve this problems with telomeres, highly repeated sequences at the ends of chromosomes

67 Figure 19-15

68 Telomeres and telomerase
Human telomeres have 100 to 1500 copies of TTAGGG at the ends of chromosomes These noncoding sequences ensure that the cell will not lose important genetic information if DNA molecules shorten during replication A polymerase called telomerase can catalyze the addition of repeats to chromosome ends

69 Telomerase function Telomerase is composed of protein and RNA
In the protozoan Tetrahymena the RNA component of the telomerase (3-AACCCC-5) is complementary to the telomere repeat sequence (5-TTGGGG-3) This enzyme-bound RNA acts as a template for adding the DNA repeat sequence to the telomere ends

70 Protecting chromosome ends
After telomeres are lengthened by telomerase, telomere capping proteins bind to the exposed 3 end to protect from degradation In many eukaryotes the 3 ends of the DNA also loop back and base-pair with the opposite strand to form a protective closed loop In multicellular organisms telomerase function is restricted to germ cells and a few other types of actively proliferating cells

71 Figure 19-16

72 Most cells have a limited life span
Telomere shortening occurs with each cell division in most cells As a result, telomere length is a counting device for how many times a cell has divided; if a cell divides too many times, telomeres could be lost Cells at risk of loss of telomeres undergo apoptosis, programmed cell death

73 DNA Damage and Repair DNA must be accurately passed on to daughter cells In addition to ensuring that replication is faithful, this also means that DNA alterations must be repaired DNA alterations, or mutations, can arise spontaneously, or through exposure to environmental agents

74 DNA Damage Can Occur Spontaneously or in Response to Mutagens
During DNA replication, some types of mutations occur through spontaneous hydrolysis reactions Depurination is loss of a purine base (A or G) Deamination is removal of a base’s amino group, changing its base-pairing properties Deamination may involve A, G, or, most often, C

75 Figure 19-17A

76 Figure 19-17B

77 DNA damage by mutagens DNA damage can be caused by mutation-causing agents, mutagens Environmental mutagens fall into two categories: chemicals and radiation Mutagenic chemicals alter DNA structure through a variety of mechanisms

78 DNA damage by chemical mutagens
Base analogs resemble nitrogenous bases and are incorporated into DNA Base-modifying agents react chemically with DNA bases to alter their structures, forming DNA adducts Intercalating agents insert themselves between adjacent bases, distorting DNA structure

79 DNA damage by radiation
Ultraviolet radiation alters DNA by triggering pyrimidine dimer formation – covalent bonds between adjacent pyrimidine bases X-rays and related types of radiation, called ionizing radiation, remove electrons from molecules, and generate highly reactive intermediates that damage DNA

80 Figure 19-17C

81 Translesion Synthesis and Excision Repair Correct Mutations Involving Abnormal Nucleotides
A variety of mechanisms are used for DNA repair Some repair takes place during replication, with specialized DNA polymerases that carry out translesion synthesis This is synthesis across regions in which DNA is damaged (e.g., DNA polymerase η synthesizes new DNA across regions containing a thymine dimer)

82 Errors remaining after DNA replication
Errors remaining after DNA replication are repaired by excision repair, in which abnormal nucleotides are removed and replaced E. coli has nearly 100 genes that code for proteins involved in this process Excision repair works by a basic three-step process

83 Excision repair Repair endonucleases are recruited to DNA by proteins that recognize damage 1. They cleave the backbone adjacent to the damage site; other enzymes remove the defective nucleotides 2. DNA polymerase (I in E. coli) replaces the missing nucleotides 3. DNA ligase seals the remaining nick in the repaired strand

84 Figure 19-18

85 Types of excision repair
Excision repair pathways are classified into two types Base excision repair corrects single damaged bases E.g., deaminated bases are detected by DNA glycosylases, which recognize and remove the base by cleaving the bond between the base and the sugar

86 Base excision repair (continued)
The sugar with the missing base is then recognized by a repair endonuclease that detects depurination It breaks the phosphodiester backbone to one side of the sugar and a second enzyme removes the sugar

87 Nucleotide excision repair
For removing pyrimidine dimers and other bulky lesions, a second type of excision repair is employed Nucleotide excision repair uses proteins that detect distortions in the DNA helix and recruit NER endonuclease (or exinuclease) that cuts the DNA backbone on either side of the lesion Helicase unwinds the DNA between the nicks, and frees it from the DNA; DNA polymerase and ligase complete the repair

88 The NER system is versatile
The nucleotide excision repair system detects and corrects many types of DNA damage Sometimes it is recruited to regions where transcription is stalled because of DNA damage; this is called transcription-coupled repair People with xeroderma pigmentosum must stay out of the sun because of mutations that prevent them from carrying out NER

89 Mismatch Repair Corrects Mutations That Involve Noncomplementary Base Pairs
Mismatch repair targets errors made during DNA replication that escape proofreading Mismatch repair is able to distinguish the original vs. the newly synthesized strand in order to correctly repair the mismatch E. coli uses a detection system based on methylation of A in the sequence GATC

90 Methylation in mismatch repair
DNA methylation does not occur immediately after DNA replication Therefore, mismatch repair systems can distinguish the original DNA (methylated) from the newly made strand (non-methylated) The incorrect nucleotide in the newly made strand is excised and replaced

91 Damage Repair Helps Explain Why DNA Contains Thymine Instead of Uracil
For some years it was not clear why DNA contained thymine instead of uracil But repair of deaminated nucleotides shows why DNA cannot contain uracil Deamination of cytosine converts it to uracil, which is detected and repaired; if DNA contained uracil normally, this type of repair could not be effected

92 Figure 19-19

93 Double-Strand DNA Breaks Are Repaired by Nonhomologous End-Joining or Homologous Recombination
Double-strand breaks cleave DNA into two fragments It is difficult for the repair system to identify and rejoin the correct broken ends without loss of nucleotides Two pathways are used: nonhomologous end- joining and homologous recombination

94 Nonhomologous end-joining
Nonhomologous end-joining uses a set of proteins that bind to ends of broken DNA fragments and join them together This is error-prone, because nucleotides can be lost from the broken ends, and there is no way to ensure the correct DNA fragments are joined

95 Homologous recombination
Homologous recombination is a more precise method for fixing double-strand breaks If the DNA molecule from one chromosome is broken, the homologue is available as a template to guide accurate repair

96 Nuclear and Cell Division
The two copies of each chromosome made during S phase are distributed into daughter cells during M phase M phase includes nuclear division (mitosis) and cytoplasmic division (cytokinesis)

97 Mitosis Is Subdivided into Prophase, Prometaphase, Metaphase, Anaphase, and Telophase
Mitosis is divided into five stages based on changing appearance and behavior of chromosomes Events during each stage are directed toward the correct distribution of one copy of each chromosome into daughter nuclei

98 Prophase After DNA replication, cells exit S phase and enter G2 phase, where final preparations are made for entry into mitosis Toward the end of G2, chromosomes begin to condense into more compact, folded structures The G2 → prophase transition is not sharply defined but cells are in prophase when individual chromosomes become visible

99 Prophase In prophase, each chromosome has two chromatids
In animal cells nucleoli disperse, but in plant cells nucleoli may still be visible The centrosomes near the nucleus, which function as microtubule organizing centers (MTOC), begin to migrate away from each other

100 Prophase (continued) As centrosomes move, they act as nucleation sites for microtubules (MTs), destined to form the mitotic spindle A dense starburst of MTs called an aster forms near each centrosome Within the centrosomes are microtubule-containing structures called centrioles

101 Figure 19-20A

102 Figure 19-21A

103 Prometaphase The onset of prometaphase is marked by the fragmentation of the membranes of the nuclear envelope Centrosomes complete their movement to opposite sides of the nucleus and the spindle MTs contact the condensed chromosomes MTs attach to chromosomes in the centromere region

104 Centromeres and kinetochores
DNA in centromeres consists of simple, tandemly repeated CEN sequences, with considerable variation among species A common feature among species is the presence of a special histone H3 called CENP-A in humans CENP-A recruits additional proteins to the centromere to form the kinetochore, to which MTs attach

105 Kinetochores Kinetochore proteins begin to assemble on centromeres shortly after S phase During prometaphase spindle MTs bind the kinetochores associated with each chromatid Forces exerted by these kinetochore microtubules gradually move chromosomes toward the center of the cell; this is called congression

106 Figure 19-22A

107 Two other types of microtubules
Polar microtubules interact with microtubules from the opposite pole of the cell Astral microtubules are shorter and form the asters at each pole Some of the astral microtubules interact with proteins lining the plasma membrane

108 Figure 19-20B

109 Figure 19-21B

110 Metaphase A cell is in metaphase when the fully condensed chromosomes are all aligned at the metaphase plate (a plane equidistant between the two poles of the spindle) Agents that interfere with spindle function (e.g., colchicine) are used to arrest cells at metaphase Examining metaphase cells allows chromosomes to be identified, generating a karyotype

111 Figure 19-20C

112 Figure 19-21C

113 Figure 19-22B

114 Figure 19-23

115 Anaphase Anaphase is the shortest phase of mitosis
The two sister chromatids of each chromosome abruptly separate and move toward opposite poles In anaphase A, the chromosomes are pulled toward spindle poles as kinetochore MTs get shorter

116 Anaphase (continued) In anaphase B the spindle poles themselves move away from each other as polar MTs lengthen Depending on the cell type, anaphase A and B may take place at the same time, or anaphase B may follow anaphase A

117 Figure 19-20D

118 Figure 19-21D

119 Figure 19-24

120 Telophase At the beginning of telophase the daughter chromosomes arrive at the poles of the spindle Chromosomes uncoil into interphase chromatin Nucleoli reappear and nuclear envelopes reform During this period, cytokinesis also takes place

121 Figure 19-20E

122 Figure 19-21E

123 The Mitotic Spindle Is Responsible for Chromosome Movements During Mitosis
The microtubule-containing apparatus responsible for separation of chromatids into daughter cells is the mitotic spindle

124 Spindle Assembly and Chromosome Attachment
Microtubules have an inherent polarity (the two ends have different chemical properties) The end where MT assembly is initiated (the centrosome in the case of the spindle) is the minus end The end where most growth occurs is the plus end

125 Figure 19-25

126 Spindle assembly During late prophase, MT growth speeds up dramatically and initiation of new MTs at centrosomes increases When the nuclear envelope disintegrates, kinetochores and MTs can come into contact When the plus end of MTs and the kinetochore bind, the MT becomes a kinetochore MT

127 Kinetochores Each kinetochore is a three-layered structure made of proteins attached to CEN sequences at the centromere The two kinetochores are located at opposite sides of a chromosome and so they usually attach to MTs from opposite spindle poles The polar MRs make contact with MTs from the opposite pole at the same time

128 Figure 19-26

129 Cells without centrosomes
Cells without centrosomes can still form spindles using a different mechanism, promoted by chromosomes This requires the involvement of Ran, the GTP-binding protein Ran binds to GTP due to a protein associated with mitotic chromosomes, and then binds importin and releases proteins that promote MT assembly

130 Chromosome Alignment and Separation
Chromosomes migrate to the central region of the spindle through a series of movements generated by pulling and pushing forces from the microtubules Chromosomes reach the central region and stay there as a result of precisely balanced forces pulling them toward opposite poles Chromatid separation requires action of topoisomerase II and changes in adhesive proteins

131 Motor Proteins and Chromosome Movement
Several motor proteins play active roles in mitosis They use energy from ATP to change shape and exert force that causes movement of attached structures Motor proteins play at least three distinct roles in movement of anaphase chromosomes

132 Roles of motor proteins in chromosome movement
1. Chromosomes are moved, kinetochores first, toward the spindle poles during anaphase A This is driven by kinesins associated with the kinetochore MTs One kinesin-like motor is at the plus end of the MT, embedded in the kinetochore, and moves the chromosome as it “chews up” the MT

133 Roles of motor proteins in chromosome movement (continued)
The other kinesin-like motor is located at the minus end of the kinetochore MTs It is embedded in the spindle pole and induces depolymerization there, “reeling in” the microtubules and the attached chromosomes

134 Roles of motors in anaphase (continued)
2. Motor proteins play a role in the movement of the spindle poles away from each other during anaphase B Bipolar kinesin motors bind to overlapping polar MTs from opposite spindle poles, forcing the spindle poles away from each other

135 Roles of motors in anaphase (continued)
3. Cytoplasmic dynein is associated with astral microtubules that are connected to the cell cortex The cell cortex is a layer of actin microfilaments lining the inner surface of the plasma membrane The dynein moves toward the minus ends of the microtubules and appears to move the spindle toward the cortex

136 Figure 19-27A

137 Figure 19-27B

138 Figure 19-27C

139 Cytokinesis Divides the Cytoplasm
After the chromosomes have separated, cytokinesis divides the cytoplasm in two It starts in late anaphase or early telophase, usually Certain cell types undergo many rounds of nuclear division without cytokinesis, forming a syncytium (multinucleate cell)

140 Cytokinesis in Animal Cells
Cytoplasmic division is called cleavage; it begins with a slight puckering of the cell surface that deepens into a cleavage furrow The furrow continues to deepen until opposite surfaces make contact and split the cell in two The furrow deepens along a plane passing through the spindle equator

141 Figure 19-28

142 Cytokinesis Signals emanating from the central part of the spindle, the spindle midzone, are important for completing cytokinesis Cleavage depends on a beltlike bundle of actin microfilaments (the contractile ring) that form just below the plasma membrane in early anaphase As cleavage progresses, the ring tightens around the cytoplasm

143 Figure 19-29A

144 Myosin and cleavage Contraction of the ring is generated by interactions between actin and the motor protein, myosin Members of Rho-GTP binding proteins regulate assembly and activation of the contractile ring RhoA is recruited to the cleavage furrow to activate proteins needed for actin polymerization, and stimulate activation of myosin

145 Figure 19-29B

146 Cytokinesis in Plant Cells
Plant cells cannot form a contractile ring because of the rigid cell wall They assemble a plasma membrane and cell wall between the two daughter nuclei In late anaphase or early prophase, a group of small vesicles from the Golgi complex align themselves across the equatorial region of the spindle

147 Cell Division Is Sometimes Asymmetric
Cytokinesis is not always symmetric; sometimes the spindle forms in asymmetric fashion This can result in one large and one small cell These occur frequently during embryonic development; sometimes cells formed in this way have differing developmental potentials

148 Figure 19-31

149 Regulation of the Cell Cycle
Variations are observed in overall length of the cell cycle relative length of time spent in various phases how closely mitosis and cytokinesis are coupled The cell cycle is regulated to meet the needs of each cell type and organism 149

150 The Length of the Cell Cycle Varies Among Different Cell Types
At one extreme are cells that divide continuously to replace cells that are constantly lost or destroyed E.g., cells involved in sperm formation, and stem cells At the other extreme are extremely slow growing tissues or even some (mature nerve or muscle cells) that do not divide at all Some cells do not divide unless stimulated 150

151 Variations in generation time
Most of the variations in generation time are based on differences in the length of G1, though S and G2 can also vary Cells that divide very slowly may spend days, months, or years in the offshoot of G1 called G0 Cells that divide very rapidly have a short G1 or may skip it entirely

152 Cell growth and the cell cycle
Although in some cases cell growth is not essential to the cell cycle, the two are generally linked A protein kinase called TOR (target of rapamycin) plays a role in the signaling network that controls cell size and coordinates it with cell cycle progression The network activates TOR in the presence of nutrients and growth factors

153 Cell growth and the cell cycle (continued)
Activated TOR stimulates molecules that control the rate of protein synthesis, leading to increased cell mass Some of the molecules also facilitate entry into S phase TOR is therefore an important regulator of both cell size and cell cycle progression

154 Progression Through the Cell Cycle Is Controlled at Several Key Transition Points
Control of the cell cycle must 1. Ensure that events of each phase are carried out in the correct order and at the appropriate time 2. Ensure that each phase is completed before the next one begins 3. Respond to external conditions

155 Key transition points At key transition points in the cell cycle, conditions in the cell determine whether or not it will proceed The first control point occurs in late G1; the G1→ S progression is called Start in yeast In animal cells, the comparable control point is called the restriction point; the ability to pass it is influenced by the presence of extracellular growth factors

156 The restriction point Cells that successfully pass the restriction point are committed to S phase Those that do not, enter into G0 and reside there until a signal allows them to reenter G1 and pass through the restriction points

157 A second transition point
Another transition point occurs at the G2  M boundary, where the commitment is made to enter mitosis In some cell types, the cell can be arrested in G2 indefinitely and the cell enters a state analogous to G0 In most cells the G1 arrest is the more prevalent type of control, but in some, such as frog eggs, the G2 arrest is more important

158 A third transition point
A third important transition point is during M phase, between metaphase and anaphase Here the commitment is made to move the two sets of chromosomes into the new cells Before cells can begin anaphase, all the chromosomes must be properly attached to the spindle

159 Figure 19-32

160 Studies Involving Cell Fusion and Cell Cycle Mutants Led to the Identification of Molecules That Control the Cell Cycle Cell fusion experiments in the 1970s gave hints about the identity of molecules that drive the cell cycle Two cultured mammalian cells were fused to form a cell with two nuclei—a heterokaryon If one cell is in S phase and the other in G1, the G1 nucleus quickly begins DNA replication, suggesting that S phase cells contain molecules that trigger the G1 → S progression

161 Figure 19-33A

162 Other observations Fusion of cells in M phase with cells in G1, S or G2 causes the interphase nuclei to immediately begin mitosis These experiments suggest that molecules in the cytoplasm drive cells from G1 to S or G2 to M Yeast cells were used to try to identify the molecules

163 Figure 19-33B

164 Temperature-sensitive cell cycle mutants in budding yeast
Yeasts carrying a temperature-sensitive mutation affecting the cell cycle can be grown at a lower (permissive) temperature Their cell cycles will be blocked at high temperatures Hartwell and colleagues identified many genes involved in cell cycle regulation using this type of mutation

165 Progression Through the Cell Cycle Is Controlled by Cyclin-Dependent Kinases (Cdks)
Phosphorylation of target proteins by protein kinases and dephosphorylation by protein phosphatases is a common mechanism for controlling the cell cycle Cell cycle progression is driven by protein kinases that are active only when bound to a cyclin These kinases are cyclin-dependent kinases (Cdks)

166 Cyclins The concentration of cyclins varies with phases of the cell cycle Mitotic cyclins are required for the G2  M transition and bind mitotic Cdks G1 cyclins are required for passage through the G1 restriction point (or Start) and the Cdks to which they bind are called G1 Cdks S cyclins are required for DNA replication

167 Mitotic Cdk-Cyclin Drives Progression Through the G2-M Transition by Phosphorylation Key Proteins Involved in the Early Stages of Mitosis Evidence of a control molecule triggering mitosis came from experiments involving frog eggs (Masui) Mature eggs develop from oocytes through meiosis; the oocyte arrests shortly after meiosis begins until a hormone signal is received Injecting the cytoplasm of a mature egg into an immature oocyte causes it to immediately proceed through meiosis

168 Figure 19-34

169 Maturation-promoting factor
Masui hypothesized that a cytoplasmic chemical that he named maturation-promoting factor (MPF) induced oocyte maturation Subsequent experiments showed that MPF triggered mitosis when injected into fertilized frog eggs Comparable molecules were soon detected in a broad range of organisms

170 MPF is a mitotic Cdk-cyclin complex
MPF was shown to be composed of a cyclin and a Cdk (a Cdk-cyclin complex) The mitotic Cdk portion of the complex is almost identical to the protein product of the yeast cdc2 gene Yeast cells with a defective cdc2 gene can function perfectly well if the human equivalent is provided to them

171 Control of Cdk-cyclin complexes
Mitotic Cdk is found consistently throughout the cell cycle It is active only when bound to mitotic cyclin and the concentration of mitotic cyclin gradually increases through G1, S, G2 until it reaches a crucial threshold at the end of G2 Halfway through mitosis, it is abruptly degraded

172 Figure 19-35

173 Activation of mitotic Cdk
Activation of mitotic Cdk involves phosphorylation and dephosphorylation The binding of mitotic cyclin to mitotic Cdk forms a cyclin-Cdk complex that is initially inactive (1) Inhibiting kinases phosphorylate two sites on the Cdk, blocking the active site (2)

174 Figure 19-36

175 Activation of mitotic Cdk (continued)
An activating phosphate is then added by an activating kinase (3) The final step is the removal of the inhibiting phosphates by a specific phosphatase enzyme (4) Once the phosphatase begins removing the phosphates, activated Cdk stimulates the phosphatase, amplifying the activation

176 Onset of mitosis Once activated the Cdk-cyclin phosphorylates lamin proteins of the nuclear lamina This causes lamina breakdown and destabilization of the nuclear envelope It also phosphorylates condensin, which is involved in chromosome condensation, and microtubule-associated proteins to facilitate assembly of the mitotic spindle

177 Figure 19-37

178 The Anaphase-Promoting Complex Coordinates Key Mitotic Events by Targeting Specific Proteins for Destruction Mitotic Cdk-cyclin phosphorylates and contributes to activation of the anaphase-promoting complex The anaphase-promoting complex functions as a ubiquitin ligase, which adds ubiquitin to proteins to target them for destruction One target is securin, an inhibitor of sister chromatid separation

179 Function of securin Prior to anaphase, sister chromatids are held together by adhesive proteins called cohesins Securin maintains this attachment by inhibiting the separase protein that would otherwise degrade the cohesins When the anaphase-promoting complex triggers destruction of securin, separase cleaves cohesin, freeing the sister chromatids

180 Figure 19-38A

181 Other activities of the anaphase-promoting complex
The anaphase-promoting complex targets mitotic cyclin for destruction, causing the kinase activity of mitotic Cdk to fall Many changes associated with exit from mitosis— cytokinesis, chromosome decondensation, nuclear envelope reassembly—depend on degradation of mitotic cyclin

182 G1 Cdk-Cyclin Regulates Progression Through the Restriction Point by Phosphorylating the Rb Protein
G1 Cdk-cyclin phosphorylates the key target Rb protein Nonphosphorylated Rb binds the E2F transcription factor Unbound E2F activates transcription of genes coding for proteins that initiate DNA replication

183 Rb and E2F While Rb remains bound to E2F, the E2F molecule is inactive and the cell cannot enter S phase But when cells are stimulated to divide by growth factors G1 Cdk-cyclin is activated, and phosphorylates Rb Rb releases E2F, which initiates transcription of genes needed for entry of S phase

184 Figure 19-39

185 Checkpoint Pathways Monitor Chromosome-to-Spindle Attachments, Completion of DNA Replication, and DNA Damage If cells proceeded from one phase of the cell cycle to the next without completing each step, daughter cells might be abnormal E.g., aneuploidy (incorrect number of chromosomes) could result Cells use a series of checkpoints that ensure each phase is completed properly before the next one begins

186 The mitotic spindle checkpoint
The mitotic spindle checkpoint prevents anaphase from beginning before the chromosomes are all attached to the spindle Kinetochores that remain unattached to microtubules produce a “wait” signal that inhibits the anaphase-promoting complex Members of the Mad and Bub protein families are involved

187 Mad and Bub One model suggests that Mad and Bub proteins accumulate at unattached kinetochores They are converted into a multiprotein complex that inhibits the anaphase-promoting complex by blocking the action of Cdc20 protein Once all the chromosomes are attached, Mad and Bud are no longer converted and the inhibition is lifted

188 Figure 19-38B

189 The DNA replication checkpoint
The DNA replication checkpoint ensures that DNA synthesis is complete before the cell exits G2 and begins mitosis Cells that are prevented from completing DNA replication fail to undergo the final dephosphorylation step in the activation of mitotic Cdk-cyclin

190 The DNA damage checkpoint
A multiple series of DNA damage checkpoints monitor DNA for damage and halt the cell cycle at various points (late G1, S, and late G2) by inhibiting different Cdk-cyclin complexes p53 protein, the “guardian of the genome,” plays a central role in these checkpoint pathways

191 Response to double-stranded DNA breaks
Breaks in DNA trigger activation of an enzyme called the ATM (ataxia telangiectasia mutated) protein kinase ATM phosphorylates checkpoint kinases, which then phosphorylate p53 (and other targets) Phosphorylated p53 is unable to bind to Mdm2; Mdm2 marks p53 for destruction

192 Response to double-stranded DNA breaks (continued)
Phosphorylated p53 is protected from degradation and activates two types of events: cell cycle arrest and cell death p53 activates the gene coding for p21, a protein that halts progression of the cell cycle by inhibiting activity of different Cdk-cyclins ATR (ATM-related) acts similarly in response to single-stranded DNA breaks

193 Figure 19-40

194 Cell death p53 stimulates production of enzymes involved in DNA repair
But if the damage cannot be repaired, p53 activates genes needed to trigger cell death by apoptosis A key protein in this pathway is called Puma (p53 upregulated modulator of apoptosis), which inactivates Bcl-2, an apoptosis inhibitor

195 Putting It All Together: The Cell Cycle Regulation Machine
The “machine” that regulates the eukaryotic cell cycle involves two interacting mechanisms 1. An autonomous clock goes through a fixed cycle over and over again via the synthesis and degradation of cyclins 2. The clock is adjusted as needed 195

196 Figure 19-41

197 Growth Factors and Cell Proliferation
In simple unicellular organisms presence of nutrients in the environment is the primary factor determining whether cells grow and divide In multicellular organisms extracellular signaling proteins, growth factors, control the rate of cell proliferation Growth factors are called mitogens

198 Stimulatory Growth Factors Activate the Ras Pathway
Mammalian cells with plenty of nutrients but no growth factors arrest in G1 Growth and division can be triggered by adding blood serum, which contains several stimulatory growth factors, such as PDGF (platelet-derived growth factor) Another is epidermal growth factor (EGF)

199 Action of growth factors
Growth factors such as PDGF and EGF act by binding receptors on the plasma membrane This activates the tyrosine kinase activity of the receptors, which triggers a complex cascade of events that ends with the cell passing the restriction point The Ras pathway plays a central role in these events

200 The Ras pathway in the cell cycle
Binding of a growth factor to its receptor leads to Ras activation (1) Activated Ras leads to phosphorylation and activation of a protein kinase called Raf, which starts a phosphorylation cascade (2) Raf phosphorylates a protein kinase called MEK, which phosphorylates a group of MAP kinases (mitogen-activated protein kinases)

201 The Ras pathway in the cell cycle (continued)
Activated MAPKs enter the nucleus and phosphorylate specific genes, including Jun and members of the Ets family of transcription factors (3) These transcription factors turn on transcription of “early genes” The early genes code for production of other transcription factors including Myc, Fos, and Jun

202 The Ras pathway in the cell cycle (continued)
Genes such as Myc, Fos, and Jun activate transcription of a family of “delayed genes” One of these encodes the E2F transcription factor, covered earlier The delayed genes include several genes coding for Cdk or cyclin molecules that form Cdk-cyclin complexes that phosphorylate Rb and trigger G1  S transition

203 Figure 19-42

204 Stimulatory Growth Factors Can Also Activate the P13K-Akt Pathway
Activated growth factor receptors may trigger other pathways besides Ras One is the PI3-kinase-Akt pathway, that begins with receptor-induced activation of phosphatidyl-inositol 3-kinase which catalyzes formation of PIP3 (phosphatidylinositol-3,4,5-trisphosphate) It leads ultimately to Akt phosphorylation and activation and suppression of apoptosis by Akt

205 Figure 19-43

206 Inhibition of cell cycle arrest
Akt inhibits cell cycle arrest through activation of a monomeric G protein called Rheb This leads to activation of TOR, a key regulator of cell growth The net effect of the PI3-kinase-Akt signaling pathway is to promote cell survival and proliferation

207 Inhibitory Growth Factors Act Through Cdk Inhibitors
Some growth factors inhibit cell proliferation, e.g., transforming growth factor  (TGF) TGFbinding to its receptor phosphorylates Smad proteins that move into the nucleus and activate expression of genes coding for proteins that inhibit proliferation Two Cdk inibitors that block cell cycle progression are p15 and p21 207

208 Apoptosis Damaged or diseased cells need to be eliminated
In such cases, the process must not damage surrounding cells Multicellular organisms accomplish this through a programmed cell death—apoptosis

209 Apoptosis and necrosis
Cell death called necrosis sometimes follows tissue injury Necrosis involves swelling and rupture of injured cells, whereas apoptosis involves a specific series of events that lead to dismantling of the cell contents

210 Steps of apoptosis The cell’s DNA segregates near the periphery of the nucleus and the cytoplasm decreases (1) The cell produces small cytoplasmic extensions and the nucleus begins to fragment (2) DNA is cleaved by an apoptosis-specific endonuclease and the cell is dismantled into small pieces called apoptotic bodies

211 Steps of apoptosis (continued)
Inactivation of a phospholipid translocator (flippase) causes accumulation of phosphatidylserine in the outer leaflet of the plasma membrane This serves as a signal for the remnants of the affected cell to be engulfed by nearby cells via phagocytosis (3)

212 Figure 19-44A

213 Figure 19-44B,C,D

214 Caspases Apoptosis proceeds through the activation of a series of enzymes called caspases They are produced as inactive precursors called procaspases and are cleaved to create active enzymes

215 Apoptosis Is Triggered by Death Signals or Withdrawal of Survival Factors
There are two main routes by which cells can activate caspases and enter apoptosis Activation can occur directly, e.g., when human cells are infected by viruses, cytotoxic T lymphocytes are activated and induce apoptosis This is triggered when cells receive cell death signals

216 Apoptosis in cell infected by viruses
Two death signals are tumor necrosis factor and CD95/Fas CD95 is a protein on the surface of infected cells; lymphocytes have a protein on their surfaces that binds CD95, causing it to aggregate Adaptor proteins attach to the CD95, which recruits procaspase-8 to the sites of clustering

217 Initiator and executioner caspases
When the procaspase is activated it acts as an initiator caspase, initiating the cascade Initiator caspases also activate an executioner caspase, caspase-3, which is important for activating many steps in apoptosis

218 The second type of apoptosis
One of the best-studied cases of the second type of apoptosis involves survival factors When survival factors are withdrawn, a cell may enter apoptosis The site of action is the mitochondrion Healthy cells have several anti-apoptotic proteins in the outer mitochondrial membrane

219 The second type of apoptosis (continued)
The proteins are structurally related to a protein called Bcl-2 which, together with other proteins, counteracts proteins that promote apoptosis (pro-apoptotic proteins) When cellular signals shift in balance toward pro-apoptotic proteins, the cell is more likely to undergo apoptosis One pro-apoptotic protein is called Bad (Bcl-2-associated death promoter)

220 Mitochondria trigger apoptosis
Mitochondria trigger apoptosis by releasing cytochrome c into the cytosol after accumulation of pro-apoptotic proteins lead to formation of channels in the outer mitochondrial membrane Cytochrome c stimulates calcium release from mitochondria and ER, where it binds IP3 receptors It also activates an initiator procaspase, procaspase-9, which then activates caspase-3

221 Damaged cells can trigger their own apoptosis
If a cell suffers such damage that it can’t repair itself, it may trigger its own demise It can enter apoptosis through the activity of p53, which acts through the protein Puma, which binds and inhibits Bcl-2

222 Figure 19-45

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