FFP1-DNA Replication 2023-24-FINAL.pptx

Royal College of Surgeons in Ireland – Medical University of Bahrain DEM YEAR 1-FFP1:DNA REPLICATION Module :FFP1 Code :FFP1 Class : MedYear1 semester 1 Lecturer : Dr Jeevan Shetty Date : 09th Oct 2023 Royal College of Surgeons in Ireland – Medical University of Bahrain Learning Outcomes • Desc...

Royal College of Surgeons in Ireland – Medical University of Bahrain DEM YEAR 1-FFP1:DNA REPLICATION Module :FFP1 Code :FFP1 Class : MedYear1 semester 1 Lecturer : Dr Jeevan Shetty Date : 09th Oct 2023 Royal College of Surgeons in Ireland – Medical University of Bahrain Learning Outcomes • Describe the process of DNA replication: Initiation, Elongation & Termination. • Explain the function of key enzymes involved in eukaryotic replication • Outline the role of Telomerase • Discuss DNA repair mechanisms DNA Replication Eukaryotes: Yeast Human cells Complicated – Many proteins involved Key process in the life cycle of cell Interest in Medicine: Protein function = target for drug design Antibiotics Cancer Target for cancer therapies Eukaryotic DNA replication Large Amount of DNA to be replicated Chromosomes are structurally complex. Disassemble nucleosomes and reassemble them in daughter strands Random distribution of old histones + delivery of new histones Numerous Proteins/Enzymes are required. Time Cell cycle:1.4hrs yeast • 16-24 hrs cultured animal cells • Human cells- 8hr to 100 days or Permanent G0 Semiconservative Replication Parental DNA Unwinding of two strands Exposed Bases Strict Watson-Crick Base Pairing Template strand 1 Parental + 1 Newly synthesized Daughter Strand Daughter Strand Introduction to genetic analysis 8th edition ©W.H. Freeman & Co. DNA replication Requirements: (1)A single-stranded template (2) Deoxyribonucleotide triphosphates (dNTPs) (of A,G,C & T) +Mg2+ (3) Replisome: Nucleoprotein Complex that co-ordinates the replication activities Numerous Enzymes and proteins (4)A primer with a free 3’ end hydroxyl group Initiation Separation of two complimentary strands occurs at ‘Origins of Replication’ Specific points where DNA replication begins Consensus sequence - short AT-rich region Eukaryotes - multiple sites From the origin, two ‘replication forks’ move outwards in opposite directions. Active synthesis requires the assembly of the replisome at the origin of replication. Each chromosome: • multiple replication origins • 1 every 3-300Kb • (1,000’s / 23 human chromosomes) Mechanism for rapidly replicating genome • • Clusters of 20-80 replicons Replication: both directions Lippincott’s Biochemistry 3rd edition © Lipponcott Williams & Wilkins What are the roles of the Proteins in the replisome? Step 1: Unwinding proteins - DNA Helicase - separate the DNA strands in an ATP- dependent process - Single-Strand Binding (SSB) proteins - bind to prevent the strands from re-associating - Topoisomerase - regulate twisting of the DNA - ‘DNA Supercoiling’ Nuclease +Ligase activity Step 2: Enzymes that replicate - Primase – Why? - DNA Polymerase • Anticancer (camptothecins), targets human type I topoisomerases. • Etoposide targets human type II topoisomerases. • Bacterial DNA gyrase is a unique target of a fluoroquinolones (e.g., DNA Polymerase Template 3’ 5’ C G A T G T G 3’ OH 5’ Primer T A C A (1) use single strand DNA as template (2) reads its template 3’ to 5’ (3) make new DNA from 5’ to 3’ (4) aligns & adds nucleotides along ss template which specifies the seq of the new chain (Watson Crick base pairing) (5) Catalyzes formation of phosphodiester bonds (DNA)n + dNTP (DNA)n+1 + Pyrophosphate (PPi) Reaction driven by subsequent hydrolysis of PPi A DNA Polymerase (1) Highly processive (≤ 1000 bases/second) PCNA - Proliferating Cell Nuclear Antigen Involved in organizing and orchestrating the replication process on both the leading and lagging strands Sliding Clamp’s Role? To encircle DNA template & keep DNA pol closely associated to the template as it rapidly moves along. (catalyses bond formation joining ≤ 1000 bases/second) (2) Proofreading activity to prevent errors • replication and • repair synthesis, • methylation, chromatin assembly and remodeling, as well as sister chromatid cohesion. • PCNA coordinates DNA metabolism with cell cycle progression by interacting with cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors. Preventing errors ? 1. Substrate specificity The DNA Pol active site can bind all four dNTP types Catalysis occurs only when the correct one is bound dNTP base pairs with the template while enzyme is in open, catalytically inactive form Enzyme conformational change with correct W-C pair = active enzyme 2. ‘Proof-reading’ : error correction activities 3’ to 5’ exonuclease activity (in the reverse direction) Removes nucleotides at the 3’ end of a new strand that are miss-matched Semi discontinuous Replication 5’ 3’ Duplex DNA’s two strands 3’ are simultaneously replicated at the replication fork But !!…….. DNA Pol can only make DNA 5’ to 3’! 5’ Result? The ‘Leading strand’ is synthesised continuously by DNA Pol travelling with the replication fork [it is ‘read’ as a template from 3’ to 5’] The “Lagging strand” is synthesised discontinuously, piece by piece 3’ 5’ How? 3’ 5’ 3’ 5’ Lagging Strand Synthesis Piece by Piece Primase makes a new primer at regular intervals Leading Strand Continuous synthesis DNA Pol Replicates the template from the primer producing a new strand in 5’ to 3’ direction DNA Pol blocked by proximity to next primer Result: a DNA strand of ~1,000bp Okazaki Fragment RNA primer http://darwin.nmsu.edu/~molbio/mcb520/mcb520images/Image1.gif • Primers removed • Gaps Filled • Backbone joined • Eukaryotic DNA Polymerases Human – multiple enzymes 3 main enzymes involved in eukaryotic replication a alpha d delta e epsilon Pol a Involved in initiating replication Associates tightly with primase to make a Pol a /primase complex 7-10nt RNA + 15dNTPs Replicates DNA by extending primer 5' to 3' No exonuclease activity - no proofreading Moderately processive Pol e & Pol d • Not associate with primase • Replicates DNA by extending primer 5' to 3’ • Highly processive - unlimited in complex with PCNA (proliferating cell nuclear antigen) • 3' to 5' Exonuclease activity • • Pol e: Leading strand synthesis Pol d: Lagging strand synthesis • Pol b: Involved in DNA repair • Pol g: Replicates Mitochondrial DNA Primer removal requires two enzymes RNA Primer DNA synthesised by Pol a DNA synthesised by Pol d or Pol e Rnase H1 Removes most of the RNA leaving one 5' ribonucleotide adjacent to the DNA Removes 5' ribonucleotide Flap endonuclease 1 DNA Pol d/e Pol a lacks proof reading FEN1 - endonuclease activity - mismatch up to 15 bp from 5' end of annealed DNA fills gaps Replication Termination in Eukaryotes Eukaryotes lack termination sequences. DNA replication proceeds until each replication fork collides with a fork from an adjacent replicon ? Problem in replicating the two ends of linear DNA strands, called the telomeres Continuous synthesis on the leading strand can proceed to the very tip of a template. But What happens at extreme end of the lagging strand? 5’ 3’ O H 3’ 5’ Primer removed but OH group available for DNA Polymerase to add nucleotide, So DNA can be replicated to the end of the strand Primer removed but no preceding Nucleotide, no OH group available for DNA Polymerase to add nucleotide Telomeres • 3’ end of each chromosome • 1,000s of tandem repeats (TTAGGG in humans) • Telomeric DNA synthesised & maintained by Telomerase = Ribonucleoprotein i.e RNA + Protein • RNA acts as template for synthesis of DNA • Adds tandem repeats to 3’ end New Template now available for Primase & Lagging strand synthesis. Telomerase activity Normal: Rapidly dividing cells, e.g., Unicellular eukaryotes Average: Human: (a)Gamete cell production – sperm (b) Germline cells During development, as cells divide and differentiate: Telomerase function declines = telomeres shorten Telomeres contain approx. 15kb hexamer repeat. After 100’s cell divisions? Chromosome ends will become damaged – genes deleted DNA damage causes cells to stop dividing and enter G0 or Apoptosis Telomerase Absence= normal senescence of somatic cells - ageing Abnormal: Enhanced activity - uncontrolled replication – Cancer Diagnostic tool Potential target for treatment DNA Repair Misincorporation errors in replication can be fatal for a cell by creating mutations The proof-reading activity of DNA Polymerase reduces the error rate from 1 in 104 - 105 to approximately 1 in 107 bases replicated The fidelity of DNA replication essential for accurate transmission of genetic information Yet errors occasionally occur requiring repair. DNA damage & repair DNA is constantly being damaged due to: • Radiation: U.V. light – fuse adjacent pyrimidines • High Energy Radiation - Double strand breaks • Chemicals: e.g. Nitrous acid – deaminates amines etc. C Uracil A A Hypoxanthine C Recognition Removal/excision Gap filling Ligation New Strand Mistake • Most damage is repaired by the cell • That which remains can cause mutations, and changes in the DNA base sequence: • Damage can be: Base-substitutions or Insertions or Deletions Mismatch repair (MMR) MUT S Mut S - Recognises mutation. 1. Occurs shortly after replication PCNA MUT L 2. Replaces mismatched bases or loops (up to 4bp) in DNA 3. Discriminate between parental & daughter strand -Methylation to GATC) Exo1 (CH3 added Defects in human MMR result in high cancer incidence 4. HNPC (Hereditary Non Polyposis Cancer) 70% mutations in MLH1 + MLH2 genes = Produce MutL proteins that carry out Mismatch repair DNA Polymerase Recruits Mut L to form tetrameric complex PCNA stimulates Mut L to make a cut in the DNA & recruits Exonuclease 1 & DNA Polymerase Exo1 removes bases from the nick past the mutation. DNA polymerase rebuilds the copy strand DNA Ligase Joins the backbone Base excision repair Replaces bases lost through chemical processes depurination or deamination DNA glycosylase: identifies & removes damaged base leaving: site apurinic or apyrimidinic AP endonuclease: recognises & cuts the backbone deoxyribose phosphate lyase: –removes the single, base-free, sugar phosphate residue. DNA Polymerase Ligase Repair Nucleotide excision repair E.Coli NER -Responds to helix distortion - Pyrimidine dimers (T-T) Exposure of a cell to UV radiation Cleaves DNA on both sides of the damaged dimer - replaces regions of damaged DNA of up to 30 bases in length helicase Defence against 2 N.B. carcinogens • Tobacco smoke • Sunlight Humans: 16 proteins Mutations affecting different proteins in the pathway were identified in two disorders: Cockayne Syndrome microcephaly, premature aging, sensitivity to sunlight, developmental delays, shortened lifespan. Xeroderma Pigmentosum Xeroderma Pigmentosum (XP) A rare human skin disease – Autosomal recessive Deficiency in nucleotide excision repair; lack of enzymes necessary for repair of DNA damage induced by ultraviolet (UV) radiation (Thymine dimers) Symptoms: - extreme sensitivity to light - skin cancer - frequent secondary tumours & associated cancerrelated death (< 30 yrs. of age) Repair of double strand breaks: 2 mechanisms Causative agents - Ionizing radiation, chemotherapeutic agents such as doxorubicin, and oxidative free radicals (1) Nonhomologous end-joining (NHEJ) The “Ku protein” = is a broken DNA sensor, that recognizes double-stranded breaks. The Ku protein holds both strands of broken DNA, leaving the ends accessible to: nucleases polymerases ligases Ends of broken DNA aligned, trimmed or filled and strands ligated NHEJ is error-prone and mutagenic associated with predisposition to cancer and immunodeficiency syndromes (2) Recombination or Homologous repair Uses enzymes and proteins that perform genetic recombination between homologous chromosomes during meiosis Uses DNA sequence information in homologous chromosome to correct the break During S phase, sister chromatid is physically close, providing a homology donor for repair Non-mutagenic NB in humans : Defects in proteins BRCA1 and BRCA2 incidence of breast, ovarian, prostate, pancreatic cancers Mutation in genes coding for BRCA1 and BRCA2 = 80% lifetime risk End Processing Strand Invasion Holiday Junction processing Repair & Ligation DNA repair – Practice questions THANK YOU Resources • Lippincott Illustrated Reviews: Biochemistry Eighth Edition-Chapter 30 • DNA Replication: • https://www.youtube.com/watch?v=TNKWgcFPHqw • https://www.youtube.com/watch?v=bee6PWUgPo8