Physiology Concepts 1.17 Cell cycle PDF

Physiology Concepts 1.17 Cell cycle Dr. Hurnik BMS 100 Week 9 Today’s Overview In-class Phases: G1, S, G2, M Checkpoints: Start transition, G2/M, metaphase-to-anaphase Checkpoint regulation Cell cycle regulation in the presence of growth factors Cell cycle regulation in the presence of unfavourable...

Physiology Concepts 1.17 Cell cycle Dr. Hurnik BMS 100 Week 9 Today’s Overview In-class Phases: G1, S, G2, M Checkpoints: Start transition, G2/M, metaphase-to-anaphase Checkpoint regulation Cell cycle regulation in the presence of growth factors Cell cycle regulation in the presence of unfavourable conditions CKIs, RB, p53 Cell survival Post-learning 1. DNA synthesis 1 2. DNA synthesis 2 3. Mitosis Cell cycle - intro The orderly sequence of events by which a cell duplicates its contents and divides in two The cell cycle is subdivided into phases or stages § We will focus on mitosis The cell cycle is driven by specific molecular signals present in the cytoplasm Cell cycle – intro continued The cell cycle consists of § 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 subphases: § § § § G1 phase: first gap S phase: synthesis G2 phase: second gap G0 phase: resting phase, postmitotic quiescent Cell cycle - visual Mitosis Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg Cell cycle phases: G1 G1: § First gap phase Preparatory growth phase prior to cell entering DNA synthesis phase Cell is metabolically active, § Many organelles are duplicated § Duration: variable (6-24hrs) § § § § Requires nutrients & growth factors RNA, protein, lipid and carbohydrate synthesis occurs § No DNA replication yet! § § Short in embryonic and cancer cells Rapid or non-existent in rapidly dividing cells Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg Cell cycle phases: S DNA and chromosomal protein synthesis occurs Duration: Lasts approximately 7-8 hours in a typical mammalian cell with a 16 hour cycle Cell is now committed to cell division Growth factors are no longer needed at this phase DNA replication occurs here, creating two identical daughter genomes Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg Cell cycle phases: G2 § G2 § Second growth phase § Interval between DNA synthesis (S phase) and mitosis (M phase) § Enzyme, protein and ATP synthesis occur § cell growth continues § Duration: lasts approximately 3 hours in a typical mammalian cell with a 16-hour cycle Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg Mitosis Cell cycle phases: M M § Mitotic phase § Cell undergoes mitosis and then cytokinesis Mitosis – covered in post-learning video 3 § Duration: 1-2 hours Anatomy and Physiology (Betts et al). Figure 3:32. Retrieved from: https://openstax.org/books/anatomy-and-physiology/pages/3-5cell-growth-and-division Top right image: Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg Cell cycle phases: G0 G0: § State of withdrawal from cell cycle § Cell is neither dividing nor preparing to divide § Instead, the cell is “doing its job” - performing it’s function within the tissue Common for differentiated cells § Examples of cells in G0: Hepatocytes, neurons Anatomy and Physiology (Betts et al). Figure 3:30. Retrieved from: https://openstax.org/books/anatomy-and-physiology/pages/3-5-cellgrowth-and-division Cell cycle progression In order to progress through the cell cycle, a variety of signals must be turned on § If the environment is not favourable or there are errors in DNA, the cell cycle can be “paused” at several main check points (aka “transitions”) Checkpoints are based on series of biochemical switches to initiate a specific cell-cycle events. § Called the cell cycle control system Features of the cell cycle control system Features of the biochemical switches § 1. Generally binary (on/off) to launch an event in a complete & irreversible fashion Why might this be important?? § 2. Robust & reliable Contains back up mechanisms to ensure efficacy under variable conditions & if some components fail § 3. Adaptable & modified to suit specific cell types Responds to specific intracellular or extracellular signals § Cyclin dependent kinases (Cdks) – more to come Cell cycle checkpoints continued Checkpoints aka “Transitions”: § Points in the eukaryotic cell division cycle where progress through the cycle can be halted until conditions are suitable for the cell to proceed to the next stage Checkpoints can be regulated by: § Factors within the cell, mostly controlled by the “health” or “state of preparation” of the cell § Factors from outside the cell – i.e. messages from other cells within the same tissue or distant cells Cell cycle Checkpoints Three major regulatory transitions at the: § 1. Start Transition (aka G1/S) § 2. G2/M transition § 3. Metaphase-to-anaphase transition (aka M-to-A) For most cells, the G1/S seems to be the ratelimiting and committing step of the cell cycle Cell cycle Checkpoints - visual G2/M checkpoint Metaphase-to-Anaphase checkpoint G1/S checkpoint Aka Start transition Adapted from: https://upload.wikimedia.org/wikiped ia/commons/3/38/0332_Cell_Cycle_ With_Cyclins_and_Checkpoints.jpg Cell cycle Checkpoints continued Specific signals MUST be present for a cell to pass through the following checkpoints § If cell detects problem inside or outside the cell it will block progression beyond the checkpoint Eg. If extracellular conditions are not appropriate for cell proliferation, central control system blocks progression through the start transition Eg. If there is a problem with completion of DNA replication, cell will be held at G2/M checkpoint Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-9. Page 968 Cell cycle control system Key to the cell cycle control system are cyclindependent kinases (Cdks) What do you suppose Cdks do biochemically? Cdks are responsible for cyclical changes in phosphorylation of intracellular proteins that initiate/ regulate the major events of the cell cycle Eg. Might activate important enzymes to DNA synthesis Cdks are controlled a group of proteins called cyclins Cyclin-Cdk complexes - intro Cyclical changes in cyclin protein levels result in the cyclic assembly and activation of cyclin–Cdk complexes at specific stages of the cell cycle. Correct & functional cyclin-cdk complexes are needed to progress through a checkpoint § Exception: M-to-A checkpoint is a little different Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-16. Page 973 Cyclin classes There are 4 classes of cyclins that form specific complexes with Cdks § 1. G1 cyclins: Cyclin D Forms complex with Cdk4 or Cdk 6 Involved in G1 phase of cell cycle, needed for initiation of transcription of G1/S cyclins to help promote passage through start transition § 2. G1/S cyclins: Cyclin E Forms complex with Cdk2 Bind Cdk’s at the end of G1 & help trigger progression through start transition Levels decrease in S phase Cyclin classes continued There are 4 classes of cyclins that form specific complexes with Cdks § 3. S-cyclins: Cyclin A Forms complex with Cdk1 and Cdk2 Bind Cdks after progression through start transition & helps stimulate chromosome duplication during S phase Levels remain elevated until mitosis; contributes to control of some early mitotic events § 4. M cyclins: Cyclin B Forms complex with Cdk1 Bind CdKs to stimulate entry into mitosis at the G2/M transition Levels decrease in mid-mitosis Cyclin-cdk complex summary There are 4 classes of cyclins that form specific complexes with Cdks Cyclin-Cdk complex Cyclin Cdk Function G1-Cdk Cyclin D Cdk4 Cdk6 Needed for progression through the start transition G1/S-Cdk Cyclin E Cdk2 Trigger progression through Start Transition S-Cdk Cyclin A Cdk2 Cdk1 Stimulate chromosome duplication Involved in early mitotic events M-Cdk Cyclin B Cdk1 Stimulate progression through G2/M checkpoint Cell cycle Checkpoints - visual G2/M checkpoint Metaphase-to-Anaphase checkpoint G1/S checkpoint Aka Start transition Adapted from: https://upload.wikimedia.org/wikiped ia/commons/3/38/0332_Cell_Cycle_ With_Cyclins_and_Checkpoints.jpg Cyclin-Cdk complex How do cyclin-Cdk complexes work? § Cyclins function by activating the Cdk FYI – another protein, cyclin activating kinase (CAK) is needed for full activation Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-12. Page 970 Cyclin-Cdk complex continued How do cyclin-Cdk complexes work? § Cyclin protein does not simply activate its Cdk partner, but also directs it to a specific target protein § Note: A cyclin-CdK complex can induce different effects at different times in the cell cycle Since accessibility of CdK substrates change during the cell cycle § Eg. Proteins the function in mitosis may only become available for phosphorylation in G2 APC/C Progression through the Metaphase-to-anaphase checkpoint is a little different § Occurs via regulated proteolysis Where have we seen this before? § A complex called the anaphase promoting complex (APC/ C; aka cyclosome) is needed Member of ubiquitin ligase family of enzymes § Used to stimulate proteolytic destruction of specific regulatory proteins § APC/C polyubiquitinates specific target proteins for destruction in proteasomes. Target proteins: securin, M-cyclins, S-cyclins Full cell cycle Let’s take a look at the full cell cycle under favourable conditions Cell cycle phases: G1 Growth factors are required in the G1 phase § Growth factors bind to specific receptors to stimulate cellular growth and proliferation Turns on early response genes and delayed response genes § Early response genes – usually transcription factors What was an example of an early response gene we have learned already? Will induce the transcription of delayed response genes. § Delayed response gene - usually Cdks, cyclins, or other proteins needed for cell division Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 Review: Ras à MapK Eg. Myc Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-47. Page 855 G1 continued In response to binding of growth factor, § Cyclin D and then E are transcribed and translated Cyclin D can form complexes with Cdk4 and Cdk 6 § Call the G1-cdk complex Cyclin E can form complexes with Cdk2 § Called the G1/S-cdk complex Active G1-cdk and G1/S-cdk complexes allows progression through the start checkpoint. G1/S checkpoint: a closer look Active G1-cdk (and G1/S-cdk) complex will target a protein called RB and phosphorylate it. § Review: G1-cdk complex is: G1/S-cdk complex is: Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 G1/S checkpoint: a closer look continued RB functions as a transcription co-repressor § Hyperphosphorylation of RB will inactivate RB § Inactive RB then releases a transcription factor E2F, allowing transcription to proceed Transcription of cyclin E => G1/S-cdk complex forms and promotes passage through the start transition Transcription of cyclin A => S-cdk complex forms Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 Putting it all together Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 Cell cycle phases: S In early S phase, cyclin D (G1-cdk complex) and E (G1/S-cdk complex) are targeted for destruction § FYI – they are targeting for destruction by a protein called SCF § How do you suppose they are targeted for destruction? This also promotes progression through the S phase of the cell cycle Cell cycle phases: S continued Active S-cdk complex allows progression through the S phase of the cell cycle § What was the S-cdk complex? § What is occurring during the S phase of the cell cycle? Cell cycle phases: G2 S-Cdk complex levels are still high in G2 M-cyclin levels begin to rise § Form a M-Cdk complex Which cyclins and cdks are part of this complex? § M-Cdk complex is needed to pass through the G2/M checkpoint At the end of G2, the S-cyclins are destroyed § Targeted for proteolysis by APC/C M-Cdk complex control Take a look at this diagram again, what do you notice about when the M-cyclins transcribed? Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-16. Page 973 M-Cdk complex control We need to be able to control the activity of Mcyclins so that mitosis doesn’t start too soon! § Once the M-Cdk complex is assembled, it is immediately inhibited via phosphorylation FYI – inhibitory phosphorylation occurs by a kinase called Wee1 § When the cell is ready for mitosis to begin, the M-Cdk complex is de-phosphorylated § FYI - de-phosphorylation occurs by a phosphatase called Cdc25 Cell progresses through the G2/M checkpoint and mitosis begins Cell cycle phases: M phase Mitosis begins § M-cdk complex is needed for activation of various proteins needed in mitosis § Mitosis proceeds: Prophase à pro-metaphase à metaphase § Before progressing to anaphase and then to telophase, we reach our final checkpoint: Metaphase-to-anaphase (M-to-A) checkpoint Molecular Biology of the Cell (Alberts et al) 6th ed. Panel17-1. Page 980 Metaphase to Anaphase checkpoint Instead of a cyclin-cdk complex being used to progress through the M-to-A checkpoint, instead we used regulated proteolysis § APC/C complex targets a protein called securin by ubiquitylation for destruction by a proteosome Securin is an inhibitory protein that protects protein linkages that hold sister-chromatid pairs together in early mitosis Destruction of securin activates a protease that separates the sister chromatids allowing progression to anaphase Molecular Biology of the Cell (Alberts et al) 6th ed. Panel17-1. Page 980 FYI – For visualization only Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-38. Page 993 Cell cycle: M phase continued After anaphase the cell continues through: § Telophase à cytokinesis Molecular Biology of the Cell (Alberts et al) 6th ed. Panel17-1. Page 980 Cell cycle: M phase continued At the end of mitosis, the M-cyclins are also targeted for destruction by APC/C § Destroying these cyclin inactivates most Cdks in cell § Then, many proteins phosphorylated by Cdks from S phase to early mitosis are dephosphorylated by various phosphatases in the anaphase cell Dephosphorylation of Cdk targets is required for the completion of M phase, including the telophase and then cytokinesis Putting it all together Fill in the table at home Phases G1 S G2 M Functions Cyclin-Cdk complexes involved Unfavourable conditions In unfavourable conditions, the cell cycle can be paused at any of the main checkpoints § Progression through G1 is delayed if: DNA is damaged by radiation, chemicals, or errors Absence of nutrients or growth factors Abnormal cell size § Entry into M is prevented when: DNA replication is not complete Presence of DNA damage Abnormal cell size § Progression through M-to-A is prevented if Chromosomes are not properly attached to mitotic spindle Let’s consider how this is done Two important molecular mechanisms: § Cdk inhibitory proteins (CKIs) § Proteins coded by tumour suppressor genes Contact inhibition CKIs Binding of Cdk inhibitory protein (CKI) § Inactivates cyclin-Cdk complex Binding stimulate rearrangement in structure of Cdk active site § Primarily used by cells to govern the activities of G1/Sand S-Cdks early in cell cycle Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-14. Page 971 CKI examples Three important CKIs are: § p16 inhibits CyclinD-cdk4 & CyclinD-cdk5 (G1-cdk complex) § p21 inhibits CyclinE-cdk2 (G1/S-cdk complex) CyclinA-cdk2 & CyclinA-cdk1 (S-cdk complex) Cyclin B-cdk1 (M-cdk complex) § p27 inhibits CyclinA-cdk2 & CyclinA-cdk1 (S-cdk complex) CyclinE-cdk2 (G1/S-cdk complex) Cyclin B-cdk1 (M-cdk complex CKIs Know: § p16 § p21 § P27 Rest are FYI Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Figure 1.19. Page27 CKIs – review at home Complex Cyclin Cdk Function G1-Cdk Cyclin D Cdk 4 Cdk6 Needed for progression through the start transition G1/S-Cdk Cyclin E Cdk2 Trigger progression through Start Transition S-Cdk Cyclin A Cdk2 Cdk1 Stimulate chromosome duplication Involved in early mitotic events M-Cdk Cyclin B Cdk1 Stimulate progression through G2/M checkpoint Associated CKI Tumour suppressor genes Let’s looks at two key tumour suppressor genes: § p53 Recognizes and binds damaged DNA Unstressed cells have lower levels of p53 since it will be bound by a protein called Mdm2 and be degraded Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-62. Page 1015 § RB Generally found in active form § Hypo- or hyperphosphorylated? Can also recognize damaged DNA Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-61. Page 1013 p53 In the presence of DNA damage, p53 will be phosphorylated, releasing Mdm2 § p53 will not be degraded Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-62. Page 1015 p53 Active p53 binds DNA and promotes the transcription of p21 § Wait… what was p21 again? p21 binds the G1/S-cdk complex, inhibiting it. § An inactive G1/S-cdk complex will pause the cell cycle at the __?__ transition Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-62. Page 1015 RB In the presence of a growth suppressor signal or DNA damage § p16 is transcribed; p16 inhibits the G1-cdk complex, which was needed to inactivate RB § RB remains activated and bound to E2F No transcription of G1/Scyclins or S-cyclins Cell cycle is paused at start transition Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Page 294 Contact inhibition Contact inhibition – The cell cycle progression can also be inhibited due to contact with: Other cells § Aka density-dependent inhibition A basement membrane or other matrix component § anchorage dependence § Most animal cells must be attached to a substratum in order to divide § This is regulated with cadherins and Beta-catenin pathway More to come next class Extracellular signaling: survival We’ve discussed how extracellular signalling can promote cell proliferation or inhibit the cell cycle § Eg. Growth factors and mitogens can promote cell growth and mitosis Cells can also receive signals to promote survival § Survival signals promote the cell cycle and prevent apoptosis PI3K-Akt-mTOR C pathway Akt in the cell cycle PI3K-Akt-mTOR C pathway § We’ve already discussed this pathway in detail, go back to your notes from a few weeks ago to review it. § Akt can promote cell cycle progression by: Akt activates/increases: § Cyclin A à activation of CDK-1 § Cyclin D à activation of CDK-4/6 Akt decreases/inactivates: § p21 and p27 Study strategies – at home Draw out the 4 stages of cell cycle § In 1 bullet point describe the main function in each stage Add in the 3 main checkpoints § Add in the specific cyclin-cdk complexes needed to get through each checkpoint § Which checkpoint is not regulated by cyclin-cdk complexes What enzyme regulates this complex? How does it work? Add in p16, p21, p27. How do they affect the cell cycle? How does RB stop the cell cycle? How does p53 stop the cell cycle? References Alberts et al. Molecular Biology of the Cell. Garland Science. Betts et al. Anatomy and Physiology (2ed). OpenStax Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Physiology Concept 1.17 Cell cycle – Post-learning 3: Mitosis Dr. Hurnik BMS 100 Week 9 Video Link https://ccnm.ca.panopto.com/Panopto/Pages/Vie wer.aspx?id=1190d973-7ceb-447b-8fd2afbb000a17c0 Today’s Overview In-class Phases: G1, S, G2, M Checkpoints: Start transition, G2/M, metaphase-to-anaphase Checkpoint regulation Cell cycle regulation in the presence of growth factors Cell cycle regulation in the presence of unfavourable conditions CKIs, RB, p53 Cell survival Post-learning 1. DNA synthesis 1 2. DNA synthesis 2 3. Mitosis Mitosis After DNA replication in the S-phase, the cell moves into the G2 phase and then the M-phase § As mentioned in class, within the M-phase, the cell undergoes Mitosis Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17.3. Page 965 Mitosis - phases Mitosis can be divided into 5 phases § Prophase § Prometaphase § Metaphase § Anaphase § Telophase & cytokinesis The end product of Mitosis is two identical daughter cells Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17.3. Page 965 Prophase Chromosomes condense § Since chromosomes have been replicated they consist of 2, closely-associated sister chromatid Mitotic spindles assemble between the two centrosomes § Centrosomes have been replicated and are being moved apart Molecular Biology of the Cell (Alberts et al) 6th ed. Panel 17-1. Page 980 Centrosome Pericentriolar matrix A centrosome is a protein organelle § Consists of a pair of centrioles surrounded by a cloud or amorphous material (called pericentriolar matrix) Preview (FYI for now): § Minus ends of microtubules are associated with the centrosome § Plus end radiates out Centrosomes undergo replication during the cell cycle in preparation for mitosis Adapted from: Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-26. Page 985 Centriole Adapted from: Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-26. Page 985 Prometaphase Nuclear envelope breaks down Chromosomes attach to spindle microtubules via a protein called a kinetochore Molecular Biology of the Cell (Alberts et al) 6th ed. Panel 17-1. Page 980 Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-30. Page 989 Metaphase Chromosomes align at the equator of the cell (halfway between the spindle poles) § Kinetochore microtubules attach sister chromatics to opposites poles of the spindle Molecular Biology of the Cell (Alberts et al) 6th ed. Panel 17-1. Page 980 Anaphase Chromatids synchronously separate forming two daughter chromosomes Kinetochore microtubules get shorter while spindle pole moves apart § Both these processes contribute to separation of chromosomes Molecular Biology of the Cell (Alberts et al) 6th ed. Panel 17-1. Page 980 Telophase & Cytokinesis Daughter chromosomes arrive at poles of spindle Chromosomes decondense and a new nuclear envelope reassembles around each set Cytokinesis § Cytoplasm divides in two forming two daughter cells Molecular Biology of the Cell (Alberts et al) 6th ed. Panel 17-1. Page 980 Mitosis: summary How would you describe the number of chromosomes in each of the following stages of the cell cycle: (n, 2n, 4n, other) § A) G1 of the cell cycle § B) Prophase § C) After cytokinesis Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-53. Page 1005 References Alberts et al. Molecular Biology of the Cell. Garland Science. Betts et al. Anatomy and Physiology (2ed). OpenStax Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Physiology Concept 1.17 Cell cycle – Post-learning 2: DNA synthesis 2 Dr. Hurnik BMS 100 Week 9 Video Link https://ccnm.ca.panopto.com/Panopto/Pages/Vie wer.aspx?id=dc0e0edd-042e-46d3-a430afbb000a174b Today’s Overview In-class Phases: G1, S, G2, M Checkpoints: Start transition, G2/M, metaphase-to-anaphase Checkpoint regulation Cell cycle regulation in the presence of growth factors Cell cycle regulation in the presence of unfavourable conditions CKIs, RB, p53 Cell survival Post-learning 1. DNA synthesis 1 2. DNA synthesis 2 3. Mitosis DNA proof-reading mechanisms Only about 1 mistake occurs for every 1010 nucleotides during DNA replication § Reminder: this is much lower than the mistake rate for transcription (RNA polymerase) DNA polymerase proofreading 1 DNA polymerase has several proofreading mechanisms: § 1. DNA polymerase activity Takes place just prior to a new nucleotide being covalently added to the growing daughter chain § Correct nucleotide has higher affinity for the DNA polymerase than an incorrect nucleotide So, energetically, incorrectly paired nucleotides are less favourable and therefore more likely to diffuse away before the DNA polymerase can add them by mistake DNA polymerase proofreading 2 DNA polymerase has several proofreading mechanisms: § 2. Exonucleolytic proofreading Occurs immediately after an incorrect nucleotide has been covalently added to the growing daughter chain § An incorrectly added nucleotide will not provide an effective 3’-OH end for DNA polymerase to add on the next nucleotide § Separate catalytic site on DNA polymerase will initiate DNA polymerase to move in the 3’ à 5’ direction, cliping off any unpaired or mispaired residues Catalytic site: 3’-to-5’ proofreading exonuclease DNA polymerase proofreading 3 DNA polymerase has several proofreading mechanisms: § 3. Strand-directed mismatch repair system This mechanism is FYI Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5-19. Page 251 Telomeres Since DNA replication occurs discontinuously on the lagging strand we end up with a shorter DNA fragment on the daughter strand once the RNA primer has been removed § Without a mechanism to deal with this problem, DNA would be lost from the end of all chromosomes each time it divides Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5-34. Page 263 Telomeres - GGGTTA Eukaryotes have specialized nucleotide sequences at the end of their chromosomes called telomeres § Many tandem repeats of GGGTTA (in humans) FYI – 1 telomere = ~1000 GGGTTA repeats Telomere & telomerase Telomere DNA sequences are recognized by telomerase § Can replenish these sequences each time a cell divides § The activity of telomerase varies based on the cell type Some cells (eg. Stem cells) have full telomerase activity Most cells have low/ minimal telomerase activity § telomeres gradually shorten until a descendant cell inherits chromosome that lack telomere function Initiates a response causing them to withdraw permanently from the cell cycle and cease dividing Called replicative cell senescence Telomerase activity Telomerase will recognize the tip of an existing telomere DNA repeat on the parent strand and elongate it in the 5’ to 3’ direction § It uses a intrinsic RNA template as a template Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5-34. Page 263 Telomerase activity Then replication of the daughter strand can be complete by the conventional DNA polymerase § The extended telomere will be used as a template for synthesis of the daughter strand Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5-34. Page 263 References Alberts et al. Molecular Biology of the Cell. Garland Science. Betts et al. Anatomy and Physiology (2ed). OpenStax Pathologic Basis of Disease(Robbins and Cotran) 10th ed. Physiology Concept 1.17 Cell cycle – Post-learning 1: DNA synthesis 1 Dr. Hurnik BMS 100 Week 9 Video Link https://ccnm.ca.panopto.com/Panopto/Pages/Vie wer.aspx?id=068ce9f7-fc26-48d7-ac6bafbb000a170a Today’s Overview In-class Phases: G1, S, G2, M Checkpoints: Start transition, G2/M, metaphase-to-anaphase Checkpoint regulation Cell cycle regulation in the presence of growth factors Cell cycle regulation in the presence of unfavourable conditions CKIs, RB, p53 Cell survival Post-learning 1. DNA synthesis 1 2. DNA synthesis 2 3. Mitosis Cell cycle - intro The orderly sequence of events by which a cell duplicates its contents and divides in two § DNA Replication occurs is what phase of the cell cycle? A) G1 B) S C) G2 D) M Adapted from: https://upload.wikimedia.org/wikipedia/commons/3/38/0332_Cell_Cycle_With_Cyclins_and_Checkpoints.jpg DNA Replication The DNA double helix acts as a template for its own duplication. § Each daughter cell will inherit a DNA double helix containing 1 original strand & 1 new strand. This is called semi-conservative replication Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5.2. Page 239 Steps of DNA synthesis DNA Replication occurs in 4 main steps: § 1. Strand separation § 2. Primer creation § 3. DNA replication § 4. Primer removal Step 1 – Strand separation If needed, review the structure of DNA § What bonds help contribute to its stability? In order for DNA synthesis to begin, the DNA double helix must the opened-up. § Two proteins are needed to open up the double helix 1. DNA Helicase Unwinds double helix What types of bond does it break? 2. Single stranded binding proteins Bind tightly and cooperatively to stabilize the single strand conformation Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5.14. Page 247 Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5.15. Page 247 Step 1 - Strand separation: replication fork Opening of the double helix creates the replication fork Adapted fromMolecular Biology of the Cell (Alberts et al) 6th ed. Figure 5.7. Page 243 At the replication fork a multienzyme complex, DNA Polymerase will be used to synthesizes both new daughter strands of DNA. § However, DNA polymerase can only add nucleotides to an existing strand of DNA § Only works in 5’ to 3’ direction Step 2 – Primer creation DNA polymerase can only add nucleotides to an existing strand of DNA. § Ie - it can only elongate a strand of nucleic acid Therefore, a primer must be build first to serve as a base-paired chain on which to add new nucleotides. § This primer is made of RNA by an enzyme called DNA primase. Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5.10. Page 245 Step 2 – Primer creation continued Since DNA can only be synthesized in the 5’ to 3’ direction, the replication fork has an asymmetric structure. § Leading strand: synthesized continuously § Lagging strand: synthesized discontinuously. The direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth RNA primer Adapted from: Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5.7. Page 243 Step 2 – Primer creation continued On the leading strand only 1 primer is required at the start of replication On the lagging strand a primer is needed for each Okazaki fragment § FYI – these primers are ~10 nucleotides long and are made at intervals of 100-200 nucleotides on the lagging strand Adapted from: Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5.11. Page 245 Step 3 – DNA replication Once the primers are built, DNA polymerase can add nucleotides in the 5’ to 3’ direction Step 4 – Replacing the primers RNA primers are removed by a DNA repair system (FYI – RNAse H) and replaced with DNA. DNA ligase then joins the 3’ end of the new DNA fragment with the 5’ end of the previous fragment Adapted from: Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5.11. Page 245 What about supercoiling? As the replication fork moves along the double-strand DNA, anything in front of the replication fork will become overwound forming supercoils. Image adapted from: Kcneuman, CC BY-SA 4.0 , via Wikimedia Commons. Steps are FYI – to guide understanding only DNA topoisomerase The enzyme DNA topoisomerase relieves the super-helical tension by breaking the phosphodiester bond. This allows the two sections of the DNA helix to rotate freely & relieve tension. The phosphodiester bond will reform as DNA topoisomerase leaves. Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5-21. DNA synthesis Summary To do at home: § Label the diagram with the enzymes or proteins needed for each step. Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 5-23. Page 254 Histones In addition DNA replication in Eukaryotes, histones must also be synthesize so that the newly replicated DNA can be packaged into nucleosomes § Histone synthesis also occurs during the S phase of the cell cycle Histone chaperones (aka chromatin assembly factors) assist formation of assembly of histone octomer & nucleosomes References Alberts et al. Molecular Biology of the Cell. Garland Science. Betts et al. Anatomy and Physiology (2ed). OpenStax Pathologic Basis of Disease(Robbins and Cotran) 10th ed.