MEDF 1012A Foundation Course Health Sciences II Overview Living Cell 2024 PDF

MEDF 1012A Foundation Course For Health Sciences II Overview Of The Living Cell II: DNA Replication, Cell Cycle and Programmed Cell Death Dr. Yeung Hang Mee, Po School of Biomedical Sciences Email: [email protected]...

MEDF 1012A Foundation Course For Health Sciences II Overview Of The Living Cell II: DNA Replication, Cell Cycle and Programmed Cell Death Dr. Yeung Hang Mee, Po School of Biomedical Sciences Email: [email protected] Office: Choh-Ming Li Basic Medical Sciences Building 6/F Room 610P 1 Learning Objectives Describe the three-dimensional characteristics of DNA and explain the stability of DNA by its structure; Understand the mechanism of DNA replication and proofreading; List the characteristics of the cell cycle and programmed cell death. 2 Transmission of Genetic Information 3  Three-dimensional Structure of DNA The stability of DNA molecule can be achieved by its specific arrangements of nitrogenous bases, deoxyribose and phosphate group. Nitrogenous bases are present inside: Advantage: reactive nitrogen species are not easily exposed. Negatively charged phosphate group and deoxyribose are present outside: Advantage: this sugar-phosphate backbone protects the bases inside it. 4 Transmission of Genetic Information 1 2 1 DNA Transcription mRNA 2 Translation Protein 5  Genetic Code During Translation– The Codon Table Each codon has three letters with combinations of G, C, A, and U. Total are 61 codons for 20 amino acids. (Why not 64 codons?) One start codon (AUG) and three stop codon for translation. Several amino acids (e.g. Leu) can be coded by many codons but some amino acids (e.g. tryptophan and methionine) can only be coded by one codon. 6  More About Codon Table Application – Human insulin for diabetes type 1 patient can be rapidly produced by bacteria E.Coli by DNA recombinant technology, why? E.Coli and human are using the same codon table. The genetic code is nearly but NOT absolutely universal. Variations exist in mitochondria (having another codon table). 7 Prokaryotic DNA Replication 8  Replication of DNA During replication, the parental DNA will become the template to copy and overall the DNA contents of cell will be double. Conservative Semi-conservative Dispersive 2 new DNA A parental DNA strand Randomly mix of parental and strands formed + a new daughter DNA strand daughter DNA strand Parents Parents Parents 1st generation 1st generation 1st generation Reference: visionlearning.com 9 *red: parental DNA; blue: newly synthesized DNA  Replication of DNA During replication, the parental DNA will become the template to copy and overall the DNA contents of cell will be double. Semi-conservative A parental DNA strand + a new daughter DNA strand Reference video: https://www.youtube.co Parents m/watch?v=4gdWOWjio BE&t=13s 1st generation Reference: visionlearning.com 10 *red: parental DNA; blue: newly synthesized DNA  Replication of Prokaryotic DNA 2 1. There are about five million base pairs in one E.Coli genome. 2. Origin (oriC): the start site of DNA replication. 3. DNA replication is a bi-directional process. 1 4. Topoisomerase: an enzyme to relieve the supercoiling produced by unwinding the parental DNA duplex (target on phosphodiester linkage). 4 unwound twisted oriC 3 Reference: Bacterial pathogen gene regulation: a DNA-structure-centred view of a protein-dominated domain Charles J. Dorman, Aoife 1C1olgan, Matthew J. DormanClinical Science Jun 01, 2016, 130 (14) 1165-1177; DOI: 10.1042/CS20160024  Replication fork After unwinding process, there are two separated DNA strands and formed like a “fork” structure. Tail of fork Two new DNA strands will be synthesized but with 1 different mechanisms: 1. Leading strand; 2 2. Lagging strand. Head of fork 12 Definitions of 5 prime (5’) end and 3 prime (3’ ) end Based on the carbon Part of double-stranded DNA number from the ribose! Reference: Lehninger – Principles of Biochemistry 7th Edition. 13  Replication of Prokaryotic DNA 1. Helicase: an enzyme to unwind the parental DNA strand (target on hydrogen bond between DNA strands). 2. Single-strand binding proteins: To prevent the strands 3 from re-associating and 2 protect them from enzymes that cleave single-stranded DNA. 1 3. DNA polymerase: To synthesize the new daughter DNA strands from 5’ end to 3’ end. Unwinding of parental strands (NEVER 3’ end to 5’ end!) 14  Replication fork Direction of DNA synthesis by DNA polymerase: from 5’ end to 3’ end. 3’ DNA polymerase Leading strand Helicase 5’ How about another strand? Head of fork 3’ Can it start synthesis from the tail of fork? DNA polymerase 5’ Tail of fork Only the 5’ end to 3’ end (blue arrow) is correct! But it takes time to open the replication fork by helicase in order to expose the 5’ end (the head of fork)! 15  Replication fork Direction of DNA synthesis by DNA polymerase: from 5’ end to 3’ end. 1 1. Once the 5’ end at the head of fork is RNA oligonucleotide exposed, a RNA oligonucleotide made by DNA primase. DNA Primase (a RNA polymerase) 2. This will lead the DNA polymerase (orange oval) to synthesize a new DNA fragment (green color) just after this RNA oligonucleotide. 2-3 5’ 4 3’ 3. This new fragment (RNA + DNA) is 3’ called Okazaki fragment. DNA polymerase 4. Later the RNA oligonucleotide will be all 5’ replaced by DNA (all in green color). 16  Replication fork Direction of DNA synthesis by DNA polymerase: from 5’ end to 3’ end. 5. At this moment a new head of fork is made by helicase (remember it takes time!) and the 2nd DNA fragment will be produced. 6. Finally, the 1st and the 2nd DNA fragments Helicase will be joined together by an enzyme called 5 DNA ligase. 5’ 2nd DNA fragment Action by DNA ligase to fill gaps 6 3’ 3’ DNA polymerase 1st DNA fragment That’s why the process is discontinuous and the strand is 5’ called “lagging” strand! 17  Examples Applications Bacterial DNA An antimicrobial target to the E.coli polymerase O157:H7 and Mycobacterium III inhibitor tuberculosis. Bacterial Ciprofloxacin (antibiotics) – target for topoisomerase gastroenteritis. inhibitor Bacterial helicase Target drug of pneumonia, urinary tract inhibitor infection and sepsis. 18  Prokaryotic DNA proofreading and repair The proofreading of DNA polymerase is based on its 3’ end to 5’ end exonuclease activity. Exo: means from the end; Nuclease: means to remove nucleotide. DNA mismatch Helicase 5’ 5’ 3’ Daughter DNA ATGAC T Parental DNA TAC TGG 3’ How can a GC or AT pair be replaced at the wrong site? DNA polymerase 5’ Addition of nucleotide Removal of nucleotide 19  Prokaryotic DNA proofreading and repair DNA mismatch 5’ 3’ Daughter DNA A T G A C T DNA structure is distorted as hydrogen 1 bonds between G and T are not the Parental DNA TAC TGG same as GC or AT. 5’ 3’ Daughter DNA A T G A C The wrong “T” nucleotide will be 2 removed by DNA polymerase (3’-5’ Parental DNA TAC TGG exonuclease activity). 5’ 3’ The correct “C” nucleotide will be added Daughter DNA A T G A C C 3 by DNA polymerase. Parental DNA TAC TGG 20 Prokaryotic DNA proofreading and repair DNA mismatch 5’ 3’ Daughter DNA A T G A C T DNA structure is distorted as hydrogen bonds between G and T are not the Parental DNA TAC TGG same as GC or AT. How the DNA polymerase can recognize the wrong “T” nucleotide from daughter DNA rather than the wrong “G” nucleotide from parental DNA? DNA mismatch Parental DNA strand is always Answer: methylated at adenine 5’ 3’ position by methylase (enzyme) Daughter DNA ATGAC T but the daughter DNA strand is not. Parental DNA TAC TGG 21 Cell Cycle 23  The Stages of Mitosis Prophase: 1. Chromosomes are condensed and nuclear envelope is broken down. 2. Microtubules are emerged from centrosomes. Pro-metaphase: 1. Microtubules are attached to the condensed chromosomes or chromatids. 2. Centrosomes are moved towards the opposite poles. 24 Source:http://philschatz.com/biologybook/resources/Figure100202.png  The Stages of Mitosis Metaphase: 1. Chromatids are lined up at the center and attached with microtubules ready for separation. Anaphase: 1. Each chromatid is now pulled towards the opposite pole. 2. The cell size is elongated. 24 Source:http://philschatz.com/biologybook/resources/Figure100202.png  The Stages of Mitosis Telophase: 1. Chromatids are surrounded by a new nuclear envelope. 2. Microtubules from centrosomes are broken down. Cytokinesis: 1. The cells are ready to separate into two individual daughter cells. 2. Cytokinesis is also known as cytoplasmic division. 25 Source:http://philschatz.com/biologybook/resources/Figure100202.png  Microtubules ⚫ They are composed of tubulin heterodimers (- and -tubulin). ⚫ They are located at the centrosome in animal cells which is associated with the nuclear membrane during the prophase stage of the mitosis. Source: The Cell: A Molecular Approach  The Stages of Cell Cycle The cell cycle of eukaryotic cells consists of four phases. 1 2 1. The 1st phase of the cell cycle, G1 (G means "gap") takes the longest time (vary from hours to years!). 2. During G1, the cell monitors its environment and its own size. In the 2nd or S phase, nuclear DNA is replicated. 27 Reference:https://www.researchgate.net/  The Stages of Cell Cycle 4 3 3. During the 3rd phase of the cell cycle G2, the cells prepare to divide, and synthesize tubulin for construction of the microtubules. 4. After G2 phase, the cells enter the 4th phase or M phase to start mitosis. 28 Reference:https://www.researchgate.net/  The Stages of Cell Cycle Cells may re-enter G1 repeatedly going through the phases of the 5 cell cycle and dividing. Resting Phase 6 5. Cells from the heart or nerves may also leave the cycle after mitosis and never to divide again. This is called resting phase G0. 6. Cells in G0 may be stimulated by growth factors to re-enter the cycle and divide e.g. liver cells. 29 Reference:https://www.researchgate.net/ Analysis of Cell Cycle by Fluorescent-activated Cell Sorter The cells are stained with 1 fluorescent dye bound with DNA so the amount of fluorescence is directly proportional to the amount of DNA in each cell. 2 1. G0/G1 phase contains non- replicated complement of DNA (x-axis: 100,000). 3 2. G2/M phase contains replicated complement of DNA (x-axis: 200,000 totally double). 3. S phase contains intermediate amount of DNA (x-axis: 110,000-180,000). 30  Factors Affecting the Cell Cycle M checkpoint is where the control system monitors the mitosis machinery. Are all chromosomes aligned? Is there any odd number of chromosomes present? G2 checkpoint is where G1 checkpoint is where the control system will the control system will initiate M phase or not. initiate S phase or not. Are all DNA contents Is cellular ATP replicated? available? 32 Reference:https://www.researchgate.net/ Central Control System of the Cell Cycle Two major groups of controlling molecules: 1. Cyclin-dependent protein kinases (Cdk) triggers the cell cycle by phosphorylating selected proteins on serine and threonine residues. 2. Cyclins bind to Cdk molecules and control their ability to phosphorylate appropriate target proteins. They undergo a cycle of synthesis and degradation in each cell cycle. 32  Central Control System of the Cell Cycle Mitotic cyclins bind to Cdk molecules during G2 and are required for the entry into mitosis. G1 cyclins bind to Cdk molecules during G1 and are required for entry into S phase. The levels of most cyclins vary in the cell cycle while the levels of the Cdk stay roughly constant. 33 Cell Senescence Cell Senescence Most normal cells in our body do not continue to proliferate forever even though they are fully nourished! Once the doubling time of dividing cells reaches the limit, the cell division slows down and finally stops and the cells enter a G0 state which they never recover. This phenomenon is known as cell senescence in aging. 35 Reference:https://www.researchgate.net/ Cell Death 35  Types of Cell Death Can you point out the differences Necrosis Apoptosis 36  Types of Cell Death Necrosis Apoptosis Cells die as a result of acute injury Cells commit suicide (active) by activating (passive) typically become swelling and a cascade of intracellular pathways. burst. The necrotic cells spill out the It is also known as programmed cell death. intracellular contents causing inflammatory responses. The apoptotic cells die without damaging its neighboring cell 37  Caspases and Procaspases This apoptotic machinery with enzymes called caspases that have a cysteine at their active sites and cleave their target proteins at specific aspartic acids. 2 Activation of other procaspases 1 1. Caspases are synthesized in 2. The active caspases activate other the cell as inactive precursors procaspases resulting in an called procaspases. amplifying the apoptotic machinery. Reference:http://individual.utoronto.ca/ 38  Caspases and Procaspases 3. Some of the activated caspases cleave other key proteins in the cell. 3 4. Some cleave the nuclear lamin* and free 3 the DNAse to cut up the DNA in the nucleus. 4 Note: The protease cascade is not reversible! *Proteins located in the peripheral region of the nucleus between Reference:http://individual.utoronto.ca/ 39 the inner nuclear envelope membrane and chromatin.  Extracellular Activation of Apoptosis 1. A protein called Fas ligand produced by lymphocytes 1 binds to the death receptor on the target cell. 2. The active death receptor 2 activates procaspase-8. 3. The active caspase-8 further activate other procaspases to proceed apoptosis. 3 40  Intracellular Activation of Apoptosis 3 2 1 1. Inside the cell, its mitochondria release the cytochrome c into cytosol. 2. Cytochrome c binds to adaptor protein called Apaf-1. 3. The complex of cytochrome c and Apaf-1 activate procaspase-9 and trigger another caspase cascade to proceed apoptosis. 41  Summary 1. The stability of DNA molecule can be achieved by its specific arrangements of nitrogenous based (inside), deoxyribose and phosphate group (outside). 2. There are 61 codons for 20 amino acids. 3. Replication of DNA is in a semi-conservative mode to pass the genetic information to our next generation. 4. Enzymes involving prokaryotic DNA replication are topoisomerase, helicase, DNA polymerase (5” end to 3” end), DNA primase and DNA ligase. 5. The proofreading of DNA polymerase is based on its 3”end to 5”end exonuclease activity. 6. CDK-dependent kinase (an enzyme) and cyclin (a protein) work with each other to control the cell cycle. 7. Apoptosis is “actively programmed” but necrosis is a passive mode of cell death. 8. Caspases are enzymes responsible for intracellular and extracellular activation of apoptosis.