Lecture 8 - The Cell Cycle PDF

Lecture 8 – the cell cycle The cell cycle - The life cycle of a cell, the period between successive divisions of a cell Cell cycle in prokaryotes - Binary fission is the normal life cycle of a bacteria cell, involves: o Replication phase (R-phase = C-period) o...

Lecture 8 – the cell cycle The cell cycle - The life cycle of a cell, the period between successive divisions of a cell Cell cycle in prokaryotes - Binary fission is the normal life cycle of a bacteria cell, involves: o Replication phase (R-phase = C-period) o Division phase (D-phase = D-period) o Interval phase (I-phase = B-period) Binary fission - Replication phase– the duration to replicate the bacterial genome (e.g. E coli 40 min), results in formation of new chromosome which has an independent point of attachment to the membrane - Division phase – segregation of daughter chromosome and other cellular components into daughter cells, is initiated by the FtsZ proteins, which assemble in form of a ring (Z- ring) at the midpoint of the cell and leads to formation of septum Binary fission – Z ring formation Binary fission – continued - Interval phase – period between division and initiation of chromosome replication 35 The cell cycle in eukaryotes The cell cycle - Interphase – G1, S and G2 phases - For a typical rapidly proliferating human cell with a total cycle time of 24 hours, the G1 phase might last about 11 hours, S phase about 8 hours, G2 about 4 hours, and M about 1 hour The cell cycle – continued - G1 phase – gap phase after the cell division, first phase in interphase, growth and biosynthesis activity phase, duration is highly variable also among different cells of the same species, cell conducts a series of checks before entering the S phase - S phase –DNA synthesis phase, starts with replication of DNA and finishes when amount of DNA in the cell is doubled - G2 – gap phase after synthesis of DNA and before cell division, cell conducts a series of checks before entering the M phase - M – mitosis phase – the cell actually divides - G0 – “resting phase”, in multicellular cells organisms most differentiated cells “exit” the cell cycle and survive for days, weeks or in some cases (e.g. nerve cells and cells of the eye lens) even the lifetime of the organism without dividing again, some G0 cells can return to cell cycle and resume replicating - These cells may be quiescient (dormant) or senescent (aging or deteriorating) - Quiescent cells may re-enter the cell cycle, senescent cells do not re-enter the cell cycle - Most somatic cells of an organism are differentiated and quiescent – they reside in the G0 phase of the cell cycle Regulation of the cell cycle in eukaryotes CDKs - The key cell cycle regulation proteins – cyclin dependent kinases (CDKs), which associate with one of different cyclins across the cell cycle to ensure accurate cell cycle progression - CDKs do not have kinase activity unless they are associated with a cyclin - Concentration of cyclins in the cell fluctuate, CDKs concentration is stable 36 CDKs – continued - genes encoding cyclins and CDKs are conserved among all eukaryotes - Each CDK can associate with different cyclins and the associated cyclin determines which proteins are phosphorylated by a particular cyclin-CDK complex - Three main cyclin-CDK complexes: G1 cyclin-CDK; S-phase cyclin-CDK; mitotic cyclin-CDK Cell cycle regulation by checkpoints - Cell cycle checkpoints are surveillance mechanisms that monitor the order, integrity and fidelity of the major events of the cell cycle: o Growth to appropriate cell size o Replication o Integrity of chromosomes o Accurate segregation at mitosis G1/restriction checkpoint - check for cell size, nutrients, growth factors, DNA damage (environmental factors), primary decision point - Cell should progress through restriction point – after growth-factor-independent cell cycle progression starts, transition to the S phase - if damage is found – G1 arrest and/or cell enters G0 phase (non-dividing state) 37 S-phase checkpoint - Is a surveillance mechanism, that responds to DNA damage (spontaneous mutations) - The stabilization of DNA replication forks, which is critical for cell survival and genome stability - A checkpoint is a cascade of signaling events that puts replication on hold until a problem is resolved G2-phase checkpoint - DNA damage checkpoint serves to prevent the cell from entering cell division (M-phase) with genomic DNA damage - highly conserved proteins that sense DNA damage and signal the cell-cycle machinery are involved - In response to extensive DNA damage, p53 activates gene expression for specific proteins to induce apoptosis M-phase checkpoint - Check for the mitotic spindle assembly - Prevents separation of the duplicated chromosomes until each chromosome is properly attached to the spindle apparatus - Arrest anaphase in case of mistake P53 protein - Nuclear DNA-binding phosphoprotein, transcription factor - able to bind to specific DNA sequences - activated by increasing the protein’s half-life and the rate of translational initiation of its mRNA - Posttranslational modification of the protein, as well as alternative splicing and binding of regulatory proteins, also are involved in activating P53 38  - pRB – key regulator of entry into cell division, acts as a tumour suppressor, promotes G0-G1 transition when phosphorylated by CDK3/cyclin-C - Mitogenic factors – factors promoting cell division Cell cycle and human pathology DNA reparation - Organisms are permanently exposed to endogenous (source of spontaneous mutations) and exogenous agents (source of induced mutations) that damage DNA - DNA repair processes exist in prokaryotic and eukaryotic organisms, many of the proteins involved have been highly conservative throughout evolution - Control of DNA repair is closely tied to regulation of cell cycle - During the cell cycle, checkpoint mechanisms ensure that a cell’s DNA is intact before DNA replication and after replication before cell division starts, failures in these checkpoints can lead to accumulation of damage 39 Mismatch repair (MMR) - Repair DNA replication errors – remove base mismatches and small insertion/deletion loops - Mismatch-repair mechanism is strand-specific – discrimination between newly synthetized strand and template Human pathology and MMR - Germ line allelic variants in the MMR genes – MLH1, MSH2, MSH6 or PMS2 – predispose to hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome - Predisposition to colorectal cancer Nucleotide excision repair (NER) - Repairs DNA damages induced by environmental factors – radiation, mutagens (including chemical) - Important mechanism in excision of UV light induced DNA damage: causes two adjacent nucleotides to stick together, distorting DNAs double-helix shape - Two repair sub-pathways exist – one for transcriptional active DNA (TC-NER) and one for global genomic (GG-NER) Human pathology and NER - Xeroderma pigmentosum - Symptoms – Skin photosensitivity, pigmentary lesions, elevated cancer risk for skin and internal cancers, skin telangiectasia (small dilated blood vessels), ocular changes and mental retardation - All XP-proteins have functions in NER and in transcription (as chromatin remodelling complex), allelic variants in germ cells lead to disease 40  - Cockayne syndrome (CS) - Incidence of CS in western Europe – 2.7 million live births - Symptoms – mental and developmental retardation, photosensitivity, progressive sensorineural hearing loss, short stature, a typical bird like face, deep-set eyes, loss of subcutaneous fat, premature ageing and progressive neurodegeneration - Defect in NER connected to mitochondrial base excision repair (BER), defective XP and CS proteins – result in defective TCR, also play role in transcription Case report - Male patient died at the age of 31,5 years with body weight of 11.3 kg - He walked unsupported at 15 months, spasticity of joints at 42 months, tremor and staggering were documented at 9 years, inability to maintain the trunk and head erect at 30 years, wheelchair-bound at 25 years - Vision became limited to light perception at 28 and one year later he was blind - CS proteins are involved in signalling of TCR-part of NER, which is the reason for the photosensitivity - CS proteins localize to mitochondria after oxidative stress → interact with mitochondrial proteins to protect the cell from oxidative stress-induced damage, in case of defect, this may contribute to the premature ageing due to excessive cell death by apoptosis The bases for cockayne syndrome Base excision repair (BER) - Remove damaged bases in DNA sequence, responsible for removing small, non-helix distorting errors - BER can repair: o Oxidized bases o Alkylated bases 41  o Deaminated bases o Inappropriately incorporated uracil o Single strand DNA breaks - BER is initiated by a DNA glycosylase that recognizes and removes the damaged base - Two pathways exist – short (removes one base lesions) and long (removes 2-20 base lesions) - No human disease is currently known to be associated with a defect in BER, which may be due to embryonic lethality Homologous recombination repair (HR) and Non-homologous end joining (NHEJ) - Both can repair double-strand breaks (DSB) - HR uses a homologous undamaged DNA template and is highly accurate - NHEJ re-joins broken ends (trims of few nucleotides before) without using a template and is often accompanied by loss of some nucleotides, (useful when sister DNA isn’t available) - Choice of mechanism depends on cell cycle stage - NHEJ being more active in G1 and HR dominating S and G2 phase Repair of DSB Human pathology involving HR and NHEJ - Defective repair of DSBs can result in chromosomal instability, which is characterized by rearrangements and loss of chromosomes - A number of human syndromes are associated with defects in DSB repair (e.g. hereditary breast/ovarian cancer) - Consequence of defects in regulation of DSB repair (e.g. in checkpoint activation) rather than due to a direct inactivation of HR or NHEJ - Ataxia telangiectasia - Symptoms – telangiectasia in the eye, progressive cerebellar degeneration, immunodeficiency, sensitivity to ionising radiation; first symptoms in childhood – problems in walking and stranding, most 42  patient are bound to wheelchairs by the age of 10 - Allelic variants in ATM gene, ATM-protein plays a role in the damage induced repair of DNA, especially double strand breaks (DSB), cell cycle regulation, in particular the G1 and S-phase checkpoints and apoptosis Cell aging and apoptosis Cell aging - Cellular senescence – irreversible arrest of cell proliferation - Senescence arrest is considered irreversible - Two main pathways are involved – p53/p21 and p16/pRB Apoptosis - Form of cell death, also known as programmed cell death is a normal phenomenon, occurring frequently in a multicellular organism - Leads to o Fragmentation of DNA o Shrinkage of cytoplasm o Membrane changes o Cell death without lysis or damage to neighbouring cells - Intracellular pathways of apoptosis – a family proteases called caspases - Caspases are synthesized in the cell as inactive precursors – procaspases, and activated by cleavage at aspartic acids by other caspases - Protease cascade is irreversible – once a cell reaches a critical point along the path to destruction, it cannot turn back After the cell death pathway is activated… - Cell shrinks and condenses - Cytoskeleton collapse - Nuclear envelope breaks down - Nuclear DNA breaks up into fragments - Cell surface is altered ➔ All: Phagocytosis by a neighbour cell or macrophage 43  - Extrinsic pathway: Initial signal comes from outside the cell, often initiated by other cells: T- Lymphocytes (have surface molecule: Fas ligand), when fas l binds to fas receptor on surface of cell → pathway initiated → caspase cascade: caspases activate each other - Intrinsic pathway: is regulated by maintaining balance of two sets of proteins in mitochondrial membrane, antiapoptotic proteins and proapopoctic proteins o healthy cell: antiapoptic bind to proapoptic proteins → blocking their action o cell damage: antiapoptotic proteins are blocked, apoptotic proteins can go into mitochondria → mitochondrial substances like cytochrome can go into cytoplasm and bind to Apaf-1 proteins to create a compound that activates caspase cascade Summary - Repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses = crucial for genome integrity - DNA lesions activate checkpoint pathways that regulate specific DNA repair mechanisms in the different phases of the cell cycle - Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired or cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis - Activation process of apoptosis is initiated by intra-/extracellular death signals - Control of cell proliferation and cell death contributes to regulation of cell numbers in multicellular organisms 44