DNA Replication PDF

= HY DNA Replication By: Jose Avila DNA and RNA Their nucleotides consist of: Base + Sugar + Phosphate Bases = Purines + Pyrimidine Purines = Adenine (A), Guanine (G) = 2 rings Pyrimidines = Thymine (T), Cytosine (C), Uracil (U only in RNA) = 1 r...

= HY DNA Replication By: Jose Avila DNA and RNA Their nucleotides consist of: Base + Sugar + Phosphate Bases = Purines + Pyrimidine Purines = Adenine (A), Guanine (G) = 2 rings Pyrimidines = Thymine (T), Cytosine (C), Uracil (U only in RNA) = 1 ring PURe As Gold rings= purines  Backbone (holding nucleotides together) Two –O groups of phosphoric acid attached to 5’ carbon react with –OH group of 3’ carbon = two ester bonds Hydrogen bonds form between bases Intermolecular force due to H being slightly positive due to bond of electronegative atom 🡪 will interact with another electronegative atom Forms double helix A – T = 2 H bonds G – C = 3 H bonds DNA replication must happen before cell division so daughter cells have right amount of DNA. Two strands of DNA act as templates for synthesis of new complementary strands 3 Proposed Mechanisms: 1. Conservative – OG DNA stays together while new DNA forms own double helix 2. Semi-conservative – Both OG strands replicate individually resulting in double helices with old and new strands 3. Dispersive – Random replication of double-old and double- new DNA Which was correct? 🡪 Meselson – Stahl Experiment 1. Replicated 15 N (heavy) only = all strands have heavy N 2. Replication Cycle 1: New 14N (light) added 1st generation = All DNA had one heavy (15N) and one light (14N) strand 3. Replication Cycle 2: Heavy and light strands separated 🡪 replicated with light N 🡪 half of DNA became light/light strands and half became light/heavy strands 4. Repeat 🡪 heavy strands reduced by half and light and strands fade Results = Semi-conservative (mixer of original and newly synthesized strand)  Origin of Replication (oriC) Each bacterial chromosome only has ONE oriC Tend to have more A-T bases, why? Easier to break 2 H- bonds Replication Fork = location of replication on chromosome Eventually meets on opposite side to complete replication Components of oriC DnaA box = site where DNA replication initiates AT-rich region = bindings of DnaA proteins to DnaA box 🡪 separation of A-T base pairs GATC methylation sites – regulatory sites for replication  Bacteria Initiation Complex DnaA protein, DiaA (DnaA-initiator-associating protein), IHF (integration host factor) OriC contains DUE, DOR with DnaA boxes and IHF binding site DNA bends around DnaA proteins Separation of AT-rich region 🡪 DnaC interacts with DnaA protein 🡪 DnaA + DnaC recruits DNA helices (DnaB protein) DNA helices moves 5’ 🡪 3’ direction breaking H- bonds Promotes movement of replication fork  DNA Helices cause supercoils in front Will hinder movement of DNA helices and replication DNA ahead of replication fork is forced to rotate in opposite directions (twisting) Topoisomerase II catalyzes reversible breakage and joining of DNA strands These breaks serve as swivels that allow two strands to rotate freely around each other = relieves coiling Inhibitors of Topoisomerase II can be used as anti-cancer Prevents replication Tumors have high levels of topoisomerase II Proteins in DNA replication Single strand binding protein- prevents strands from coming back together Primase – required to synthesize short RNA chain Complementary to DNA strand and forms DNA-RNA (has U) Eventually removed and replaced with DNA Needed because DNA polymerase can only ADD to growing polymer DNA Polymerase I – removes RNA primer and replaces with DNA DNA Polymerase III – main enzyme complex 10 subunits = DNA polymerase III holoenzyme DNA Polymerase III Attaches NTs at 3’ end Have 3 phosphates attached = dNTP, deoxynucleotide triphosphate First phosphate from 5’ C of incoming dNTP with –OH group of 3’ of deoxyribose Energy released from separation of pyrophosphate 2 unreacted phosphates of dNTP Drives formation of ester bonds between template strand 3’ C and 5’ phosphate of incoming NT  Leading VS. Lagging Strand Strand DNA pol. III moves 3’ 🡪 5’ and DNA pol. III moves on synthesizes 5’ 🡪 3’ template (parental strand) in 3’ Replication fork moves in same direction – so polymerase must make short stretches 🡪 5’ of new DNA 5’ 🡪 3’ (Okazaki fragments) New strand synthesis from 5’ DNA polymerase I excises the RNA primers and fills in with DNA 🡪 3’ = leading strand DNA ligase catalyzes phosphodiester bonds linking the Okazaki fragments together Many primers are needed along the lagging strand Allows bidirectional replication Enzyme complex moves in one direction synthesizing both strands and this is why lagging strands are composed of Okazaki fragments Mutations + Proofreading Mistakes can lead to mutations = functional alteration / disease Polymerase use its proofreading ability to cleave phosphodiester bond of improper NT then add correct one Regulation of Bacterial DNA Replication Occurs by regulating initiation of replication at oriC by 2 mechanism: DnaA proteins regulated using ATP DnaA-ATP bind to DnaA boxes to initiate AT-rich region separation Following replication – the new copy of DNA has doubled the DnaA boxes When DnaA-ATP binds to DnaA boxes the ATP is hydrolyzed to ADP → DnaA-ADP has a low affinity for DnaA boxes Only when a cell is ready for division does the DnaA-ATP reach sufficient levels to promote one round of DNA replication GATC methylation GATC nucleotide sequences are found within the region of oriC When a cell is ready to undergo cell division – the enzyme DNA adenine methyltransferase (Dam) is activated Dam adds –CH3 groups to the A of the GATC sequence (so adenines become methyladenines) Methyladenines aid in recruiting the enzymes of replication to the oriC When replication occurs – DNA polymerase III adds adenines to the daughter strands and not methyladenines thus preventing a second round of replication Termination Ter sequences (T1 and T2) – on bacterial chromosome opposite side of oriC T1 🡪 stops left to right movement T2 🡪 stops right to left movement Tus (termination utilization substance) is bound to ter sequences T1 and T2 near each other DNA ligase covalently links 2 daughter strands creating 2 circular double- stranded DNA molecule Eukaryotic DNA Replication Initiation: MCM2-7 Minichromosome maintenance helicases Origin Recognition Complex (ORC) - markers on DNA that recruit replication forks Cdc6 and Cdt1 associate with Ori sites and recruit MCM2-7 helicases MCM + Cdc6 + cdt1 = prereplication complex (pre-RC) CDK2-cyclin E and Cdc7-Dbf4 (DDK kinases) initiates origin firing by multiple phosphorylation events leads to replisome assembly Cdc6 and Cdt1 no longer required and removed MCMs and GINS and Cdc45 unwind DNA to expose template DNA – replisome assembly can be completed and replication initiated  Eukaryotic DNA Polymerases DNA polymerases α (alpha), δ (delta), ε (epsilon) = involved in nuclear DNA replication DNA polymerase γ (gamma) = involved in replication of mitochondrial DNA Also requires primase: DNA pol. α associates with primase and produces RNA primer of 10 NTs then stretch of DNA 20-30 Nts DNA polymerase δ and ε complete leading and lagging strand syntheses building of primer and short DNA pieces Synthesize in 5’ 🡪 3’ only Cannot covalently link together first 2 ind. NTs Only replicate preexisting strands (Or catalyze primers) No primers can be synthesized upstream from 3’ end – thus chromosome becomes progressively shorter with each replication (or cell division )  Termination in Eukaryotics DNA pol. Stops when it reaches section already replicated for (due to multiple origins of replication) Will dissociates because cannot form phosphodiester bond FEN1 (flap endonuclease 1) and Rnase H remove RNA primers at start of each leading strand and start of each Okazaki fragments DNA ligase creates phosphodiester bond between unconnect sugar-phosphates to create long continuous DNA strand (makes it look pretty and put together)  Telomeres: Additional sequences at 3’ end, composed of complex repeat sequences (TTAGGG) and 3’ overhang region of DNA (12-16 NTs) Prevent genetic information loss from chromosome shortening Telomerase – recognizes ends of chromosomes and synthesizes telomeres Composted of protein and RNA subunits  Short 3-nucleotide RNA sequence of the telomerase allows it to bind to the 3’ end of the DNA overhang Adjacent 6-nucleotide RNA sequence of the telomerase is used as the template to make a 6-nucleotide repeat of DNA – process is catalyzed by two subunits called telomerase reverse transcriptase After the repeat is completed, the telomerase moves 6 nucleotides to the right and synthesizes another repeat, then the cycle is repeated many times (1,000-2,000 copies) DNA polymerase synthesizes a complementary strand to the telomeric repeats adding to an RNA primer Cell Cycle Identifying Mitosis Stages Cell Cycle Repeated pattern of growth, DNA duplication and cell division that occurs in eukaryotic cells (avg = 16h) Purposes = Growth and Repair 3 phases: Interphase = cell growth Mitosis = division of nuclear material 🡪 2 daughter cells Cytokinesis = division of cytoplasmic material Checkpoints in Cell Cycle CDKs and cyclins bind 🡪 CDK initiates cell cycle events while cyclins help direct CDKs to correct targets Cyclin CDK Cyclin-CDK Action S CDK2 S-CDK Promotes DNA replication initiation M CDK1 M-CDK Promotes Mitosis G1 CDK4 or CDK6 G1-CDK Promotes progression of G1 🡪 S G1/S CDK2 G1/S-CDK Commit to DNA replication (S phase)  Inhibit phosphatase MORE (Cdc25) to prevent Inhibit action of APC REGULATION CDKs dephosphorylated by phosphatase = CDK activation ON Removing Pi CDKs phosphorylated by kinases = OFF Adding phosphate Inhibit CDK activation (active Wee1)  G1/ G0 Phase Decision by cell: First Checkpoint NOT proliferating 🡪 G0 Cell proliferating 🡪 G1 Mitogens – factors that promote cell signaling to activate cell cycle Lack of mitogens 🡪 cell arrest in G0 Can activate cells from G0 🡪 G1 If removed, cells can arrest in G1 for a period Mitogens stimulate proliferation (into G1) by inhibiting Rb protein Rb missing causes retinoblastoma (childhood eye tumor Mitogens = cyclin-CDK proteins Rb binds and inhibits E2F (transcription factor) G1-CDK phosphorylates Rb 🡪 releases E2F 🡪 E2F binds promoter to upregulate self and cyclin E Cyclin E = G1/S-SDK Proteins involved in mitotic spindle formation G0 and beyond G0 = Quiescence Nondividing state, unfavorable conditions for growth GTD = Senescence Permanent state of quiescence Terminally differentiated Apoptosis – cell death Conflicting signals promote cell death Restriction Point (R Point) G1 committed to beginning replication cycle S phase S-CDK activated at end of G1 and promotes DNA replication Pre-replication complex binds to origin of replication in DNA in G1 S-CDK phosphorylates/activates helicase S-CDK aids assembly of replication fork DNA pol. binds and replicates  Summary Interphase: G1-S-G2) Before mitosis – DNA must be replicated and centrosome duplicated to form two poles of the cell Centrosome = microtubule organizing center (MTOC) Duplication of centrosomes occurs during DNA replication (triggered by S-CDK and G1/S-CDK) Mitosis – activated by M- CDK M-CDK activated by Cdc25 🡪 mitotic spindle construction and prepares duplicated chromosomes for segregation Positive feedback Prophase: Prometaphase Metaphase Anaphase Telophase 1. Duplicated 1. Nuclear membrane 1. Chromosomes 1. Sister chromatids 1. Chromosomes chromosomes broken into small align at equator of separated arrive at poles condense vesicles mitotic spindle 2. Kinetochore 2. Nuclear envelope 2. 2 centrosomes 2. Chromosomes microtubules get reassembles separate and each attach to mitotic shorter and “nucleate” arrays of spindles via spindle poles End Mitosis microtubules (asters) kinetochores of move apart chromosomes Prophase Chromosome + its copy = sister chromatids Cohesins added along sister chromatids To reduce/compact large chromosomes, as cell enters M phase condensins coil each sister chromatids Microtubule nucleation and asters Mitotic Spindle formation Microtubules growing from one centrosome interact with microtubules from other centrosome Interaction stabilizes microtubules and prevents depolymerization Prometaphase Nuclear envelope disassembles Lamins phosphorylated 🡪 fragmentations Nuclear pore complexes disassemble Motor proteins organize envelope fragments (come back later) Kinetochores attach chromosomes to mitotic spindle Are protein complexes that assemble at centromere of chromosomes One per sister chromatid and face opposite direction – binds to microtubules from opposite poles Regulation: lack of tension = mitosis inhibited (indicated unattached sister chromatid Metaphase Duplicated chromosomes move around via tension on mitotic spindle 🡪 align along metaphase plate Indicated beginning of metaphase  Mitotic Checkpoint: during metaphase Unattached/ improperly attached kinetochores = checkpoint active Activation site for MAD2 (mitotic arrest deficient 2) 🡪 MAD2* MAD2* inhibits anaphase onset by blocking Cdc20-APC BubR1 synergizes with MAD2* Attached properly = Checkpoint inactive MAD2 NOT activated BubR1 does NOT interact with Cdc20-APC Cdc20-APC 🡪 cyclin B degradation leads to CDK1 inactivation 🡪 mitosis to cytokinesis Securtin bound to separin – degradation of securing releases separin to cleave ring of protein holding sister chromatids together 🡪 onset of anaphase  Spindle Assembly Checkpoint 1. Lack of tension on 1.Tension applied to kinetochore kinetochore 2. Aurora kinase active 2.Aurora kinase inactive 3. Phosphorylates MAD2 3.MAD2 inactive (mitotic arrest deficient 2) 4.APC activated 4. MAD2 prevents APC activation  Anaphase APC triggers chromosome separation APC ubiquitinates M cyclin 🡪 M cyclin proteolyzed APC ubiquitinates securin 🡪 separase activated 🡪 separase cleaves ring structure of cohesins Mitotic spindles can now pull sister chromatids apart All chromosomes released at same speed to respective poles Loss of αβ-tubulin from both ends of kinetochore microtubules Motor proteins Kinesin sliding interpolar microtubules past one another at equator [(-) 🡪 (+)] Dynein anchored at cell membrane of poles pulling on pole itself [(+) 🡪 (-)] Telophase Nucleus reconstruction Mitotic spindles dissemble Nuclear membrane reassembles around each group of chromosomes 🡪 forms nuclei Vesicles of nuclear membrane cluster around chromosomes and fuse to form nuclear envelope Lamins dephosphorylated and reassemble scaffolding of nuclear (with aid of motor proteins) Cytokinesis – cytoplasm cleaved in 2 producing 2 separate daughter cells Chromosomes arrive at poles and nuclear envelope reassembled First initiated during anaphase Utilizes actin and myosin filaments 🡪 together make up contractile ring Cleavage furrow develops because of contractile ring 🡪 develops perpendicular to long axis of mitotic spindle Overview of Cell Cycle 2 irreversible points 1. Replication of genetic material 2. Separation of sister chromatids Checkpoints = critical control points in cell cycle where “stop” and “go” signals can regulate progression 3 major: G1, G2, and M phases  Aneuploidy and Human Solid Tumors Aneuploidy = condition of having an abnormal number of chromosomes in a haploid set. Mitotic checkpoint prevent chromosome missegregation and aneuploidy Altered expression level of mitotic checkpoint proteins occur commonly in tumors Reduction in checkpoints leads to near-diploid aneuploidy from nondisjunction errors 🡪 both copies of one or few chromosomes deposited in same daughter cell Complete inactivation of the mitotic checkpoint pathway results from elimination of key components such as MAD2 or BubR1 – massive chromosome missegregation  Meiosis and Reproduction Meiosis = formation of gametes (sexual reproduction cells)”🡪 only have one-half of chromosomes found in body cell Body cell (whole set) = diploid Gamete cell (half-set) = haploid 2 types of gametes: Sperm (males) Eggs / ova (females) Gametogenesis: Spermatogenesis 🡪 4 haploid sperm cells Oogenesis 🡪 1 ovum or egg cell Fertilization of egg with sperm restores chromosome number to 2n Meiosis vs. Mitosis 8 phases. vs. 4 Cell divides twice vs. once  Phases of Meiosis: 1. Prophase I – longest phase, chromosomes condense Synapsis- homologous chrom. Pair up = tetrad Crossing over occurs 2. Metaphase I- shortest phase, homologous pairs align Independent assortment occurs –adds variation 3. Anaphase I- Tetrads separate (homologous chromosomes separate) Sister chromatids remain attached 4. Telophase I and Cytokinesis I- each pole has haploid set 🡪 2 haploid daughter cells 5. Prophase II 6. Metaphase II 7. Anaphase II 8. Telophase II and Cytokinesis II – same as DNA not replicated again b/w mitosis, nuclei form Meiosis I and Meiosis II 4 haploid daughter cells produced (single Meiosis II is similar to mitosis  Prophase I –Crossing Over Crossing over may occur in tetrad between non-sister chromatids, ends break and reattach Provides variation in genetic material Metaphase- Independent Assortment Tetrads can line up two different ways before homologs separate Also, allows more variation in genetic material Nondisjunction – tetrad (anaphase I) or sister chromatids (anaphase II) do not separate Creates abnormal number of chromosomes in gametes Fatal most of time Jose Avila [email protected] 610-533-8825