DNA Replication - EJMG Oct 2024 PDF

http://zarkauthor.typepad.com/.a/6a00e5500940a988340134805a3dac970c-500wi DNA REPLICATION  DNA Replication Types of DNA replication Semi-conservative model of DNA replication Prokaryotic DNA r...

http://zarkauthor.typepad.com/.a/6a00e5500940a988340134805a3dac970c-500wi DNA REPLICATION  DNA Replication Types of DNA replication Semi-conservative model of DNA replication Prokaryotic DNA replication Eukaryotic DNA replication Inhibitors of DNA replication (Analogues, Intercalation, Polymerase Inhibitors) DNA damage Types and agents of mutations Spontaneous, Radiation, Chemicals. Repair mechanisms Base Excision, Nucleotide Excision, Mismatch Repair. DNA-recombination In meiosis Transposition  Central dogma Replication Transcription Translation DNA RNA PROTEIN Reverse transcription  DNA replication is a biological process that occurs in all living organisms and copies their exact DNA. It is the basis for biological inheritance. Replication is the process of synthesis of daughter DNA from parental DNA by the enzyme DNA Polymerase. ( dNMP )n + dNTP ( dNMP )n+1+ PPi DNA Lengthened DNA DNA Replication Parental strand Daughter stand  DNA Replication  A reaction in which daughter DNAs are synthesized using the parental DNAs as the template.  Transferring the genetic information to the descendant generation with a high fidelity. Replication Parental DNA Daughter DNA 6 Three possible replication patterns: 1. Semiconservative replication 2. Conservative replication 3. Dispersive replication Semiconservative replication Conservative replication Dispersive replication  Each parent strand serves as a template for a new strand and the two new DNA Semiconservative replication strands each have one old and one new strand http://room114.wikispaces.com/file/view/DNARepAnima4.gif/31922479/DNARepAnima4.gif http://vce.bioninja.com.au/_Media/semi_conservative_med.jpeg Parent strands New / Daughter strand Characteristics of Replication ⚫ Semi-conservative replication ⚫ Bidirectional replication ⚫ Semi-continuous replication ⚫ High fidelity 10 Meselson and Stahl experiment demonstrated semiconservative replication  5’ 3’ Identical 5’ base sequences 3’ 3’ 5’ 3’ 5’ Semiconservative Replication Half of the parental DNA molecule is conserved in each new double helix, paired with a newly synthesized complementary strand. This is called semiconservative replication. Direction of the DNA Replication  Bidirectional Replication Replication starts from unwinding the dsDNA at a particular point (called origin / ori site), followed by the synthesis on each strand. The parental dsDNA and two newly formed dsDNA form a Y-shape structure called Replication fork. Replication of Prokaryotes The replication process starts from the origin, and proceeds in two opposite directions. It is named - Replication. 21  Replication Enzymes & Proteins  DNA Polymerase - Matches the correct nucleotides then joins / polymerizes adjacent nucleotides to each other.  Helicase - Unwinds the DNA and melts it.  Primase - Provides an RNA primer to start polymerization.  Single Strand Binding Proteins - Keep the DNA single stranded after it has been melted by helicase  Gyrase - A topisomerase that Relieves torsional strain in the DNA molecule. Ligase - Joins adjacent DNA strands together (fixes “nicks”)  Telomerase - Finishes off the ends of DNA strands in Eukaryotes DNA REPLICATION  Enzymes and proteins of DNA Replication Protein MrW Sub Function units Dna A protein 50,000 1 Recognizes ori sequences Dna B protein 300,000 6 Unwinds/opens dsDNA (DNA Helicase) Dna C protein 29,000 1 Assists Dna B to bind at ori-site DNA polymerases Synthesizes the new DNA strands Dna G protein 60,000 1 Synthesize RNA primer (DNA Primase) Single Strand Binding 75,600 4 Binds single-stranded Proteins (SSB) DNA DNA Gyrase 400,000 4 Relieves torsional strain (DNA Topoisomerse) generated by unwinding  DNA Polymerases of Prokaryotes DNA Polymerase-I  The first DNA- dependent DNA polymerase ( DNA Pol -I ) was discovered in 1958 by Arthur Kornberg who received Nobel Prize in physiology & medicine in 1959.  DNA Polymerase is considered as Kornberg Enzyme.  Later, DNA-Pol II and DNA-Pol III were identified.  All of them possess the following biological activity. 1. 5→3 Polymerse activity 2. Exonuclease activity Comparison of DNA Polymerases of E. coli  Exonuclease functions 5´→3´ 3´→5´ exonuclease exonuclease activity activity excise mismatched removes primer or nuleotides excise mutated segment 5' 3' C T T C A G G A ？ G A A G T C C G G C G 3' 5'  DNA Polmerase - I  Mainly responsible for proofreading and filling the gaps, repairing DNA damage 30  DNA Polymerase - II  Temporarily functional when DNA-pol I and DNA-pol III are not functional.  Still capable for doing synthesis on the damaged template.  Participates in DNA repair process.  DNA Polymerase - III  A heterodimer enzyme composed of ten different subunits  Having the highest polymerization activity (105 nt/min)  The true enzyme responsible for the elongation process  Structure of DNA-pol III α： has 5´→ 3´ polymerizing activity ε：has 3´→ 5´ exonuclease activity and plays a key role to ensure the replication fidelity. θ: maintain heterodimer structure Nucleotides are always added to the growing strand at the 3’ end – the end at which the DNA strand has a free –OH group on the 3’ carbon of its terminal deoxyribose Free 3’- hydroxyl group  RNA Primase Also called DnaG Primase is able to synthesize primers using free NTPs as the substrate and the ssDNA as the template. Primers are short RNA fragments of a several nucleotides long.  Primers provide free 3´-OH groups to react with the -P atom of dNTP to form phosphodiester bonds. Primase, DnaB, DnaC and an origin form a Primosome complex at the initiation phase.  Helicase Also referred to as DnaB. It opens the double strand DNA with consuming ATP. The opening process with the assistance of DnaA and DnaC  SSB protein Single Strand DNA Binding protein. SSB protein maintains the DNA template in the single strand form in order to prevent the dsDNA formation; protect the vulnerable ssDNA from nucleases.  DNA Gyrase It cuts phosphoester bonds on both strands of dsDNA, releases the supercoil constraint, and reforms the phosphodiester bonds. It can change dsDNA into the negative supercoil state with consumption of ATP.  DNA Ligase 3' 5' 5' 3' RNAase 3' 5' 5' OH P 3' dNTP DNA polymerase 3' 5' 5' P 3' ATP DNA ligase 3' 5' 5' 3'  Connect two adjacent ssDNA strands by joining the 3´-OH of one DNA strand to the 5´-P of another DNA strand. Sealing the nick in the process of Replication, Repairing, Recombination, and Splicing.  Replication Fidelity Replication based on the principle of base pairing is crucial to the high accuracy of the genetic information transfer. Enzymes use two mechanisms to ensure the replication fidelity. – Proofreading and real-time correction – Base selection  Proofreading and Correction  DNA-pol I has the function to correct the mismatched nucleotides.  It identifies the mismatched nucleotide, removes it using the 3´- 5´ exonuclease activity, add a correct base, and continues the replication. DNA Replication Process DNA REPLICATION STAGES Initiation Elongation Termination  Three Stages of replication 1). Initiation ✓ occurs at the origin of replication ✓ separates dsDNA, primer synthesis 2). Elongation ✓ involves the addition of new nucleotides (dNTPs ) based on complementarity of the template strand ✓ forms phosphoester bonds, correct the mismatch bases, extending the DNA strand, … 3). Termination ✓ stops the DNA Replication occurs at a specific termination site  Genome of E. coli ori-Site  Initiation  The replication starts at a particular point called origin of Replication (or) ori-Site.  The structure of the origin is 248 bp long and AT-rich. 13 mer- sequence 9 mer- sequence Formation of Preprimosome  Origin of Replication Site where DNA synthesis starts  Formation of Replication fork  DnaA recognizes ori C.  DnaB ( Helicase ) and DnaC join the DNA- DnaA complex, open the local AT-rich region, and move on the template downstream further to separate enough space.  DnaA is replaced gradually.  SSB protein binds the complex to stabilize ssDNA.  Primer synthesis  Primase joins and forms a complex called primosome.  Primase starts the synthesis of primers on the ssDNA template using NTP as the substrates in the 5´- 3´ direction at the expense of ATP.  The short RNA fragments provide free 3´-OH groups for DNA elongation.  INITIATION Primase synthesizes PRIMER 3’ Primer 5’ Single stranded binding protein 5’ 28-10-24 12:42:53  Elongation  dNTPs are continuously connected to the primer or the nascent DNA chain by DNA-pol III.  The core enzymes (、、and  ) catalyze the synthesis of leading and lagging strands, respectively.  The nature of the chain elongation is the series formation of the phosphodiester bonds.  ELONGATION 3’ Primer 5’ Leading strand Primer is removed Elongation byPolymerase by DNA DNA Polymerase III I Parental DNA 5’ 3’ Okazaki fragments 5’ 3’ 5’ 3’ Laging strand Gap filled byDNA Ligase 5’ 28-10-24 12:42:53  Lagging strand synthesis  RNA Primers on Okazaki fragments are digested by the enzyme RNase.  The gaps are filled by DNA-pol I in the 5´→3´direction.  The nick between the 5´end of one fragment and the 3´end of the next fragment is sealed by ligase.  Okazaki fragments Many DNA fragments are synthesized sequentially on the DNA template strand having the 5´- end. These DNA fragments are called Okazaki fragments. They are 1000 – 2000 nt long in prokaryotes and 100-150 nt long in eukaryotes. The daughter strand consisting of Okazaki fragments is called the lagging strand. 64 Directionality of the DNA strands at a replication fork Lagging strand Leading strand Fork movement  Lagging strand (discontinuous) Leading strand (continuous) 3' 5' 5' 3' RNAase 3' 5' 5' OH P 3' dNTP DNA polymerase 3' 5' 5' P 3' ATP DNA ligase 3' 5' 5' 3'  Termination  The replication of E. coli is bidirectional from one origin, and the two replication forks must meet at one point called ter at 32.  All the primers will be removed, and all the fragments will be connected by DNA-pol I and ligase. Ter-binding proteins - will recognizes the Termination sequences and helps to achieve the termination process. End of replication INHIBITORS OF DNA REPLICATION Nalidixic acid Novobiocin Ciprofloxacin INHIBITORS OF DNA REPLICATION Adriyamycin Etoposide Doxorubicin Eukaryotic DNA Replication  DNA replication is closely related with cell cycle. Multiple origins on one chromosome, and replications are activated in a sequential order rather than simultaneously.  DNA Polymerse of Eukaryotes Eukaryotic Enzyme Prokakaryotic Enzyme DNA-pol : initiate replication DnaG, and synthesize primers primase DNA-pol : replication with Repair low fidelity DNA-pol : Mitochondrial DNA synthesis DNA-pol : elongation DNA-pol III DNA-pol : lagging strand DNA-pol I synthesis, proofreading and gap filling  Initiation  The eukaryotic replication origins are shorter than that of E. coli. The ori-sites in Eukaryotes called ARS (Autonomously Replicating Sequences) (or) Replicators.  Requires DNA-pol  (primase activity) and DNA- pol  (polymerase activity and helicase activity).  DNA-pol  requires a protein called for its activity Proliferating Cell Nuclear Antigen (PCNA).  Needs Topoisomerase and Replication factors (RF) to assist.  Elongation  DNA replication and nucleosome assembling occur simultaneously.  Overall replication speed is compatible with that of prokaryotes.  Termination 3' 5' 5' 3' 3' 5' 5' 3' connection of discontinuous segment 3' 5' 5' 3' 3' 5' 5' 3' 83  Telomere  The terminal structure of eukaryotic DNA of chromosomes is called telomere.  Telomere is composed of terminal DNA sequence and protein.  The sequence of typical telomeres is rich in T and G.  The telomere structure is crucial to keep the termini of chromosomes in the cell from becoming entangled and sticking to each other. 84  Telomerase The eukaryotic cells use telomerase to maintain the integrity of DNA telomere. The telomerase is composed of telomerase RNA telomerase association protein telomerase reverse transcriptase It is able to synthesize DNA using RNA as the template. 85  Step in Replication Prokaryotic cells Eukaryotic cells Recognition of origin of Dna A protein RpA replication (Replication Protein-A) Unwinding of DNA double helix Helicase Helicase (requires ATP) (requires ATP) Stabilization of unwound Single-stranded DNA-binding Single-stranded DNA-binding template strands protein (SSB) protein (SSB) Synthesis of RNA primers Primase Primase Synthesis of DNA Leading strand DNA polymerase III DNA polymerase δ Lagging strand DNA polymerase III DNA polymerase Ԑ Removal of RNA primers DNA polymerase I RNAse-H (5 →3' exonuclease) Replacement of RNA with DNA DNA polymerase I Unknown Joining of Okazaki fragments DNA ligase DNA ligase (requires NAD) (requires ATP) Removal of positive supercoils DNA topoisomerase II DNA topoisomerase II ahead of advancing (DNA gyrase) replication forks  BASE ANALOGUES A base analog is chemical that can substitute for a normal nitrogen base in Nucleic acids. They are categorized in two separate groups, purine analogues and pyrimidine analogues. Oncologists employ 5-fluoro- or 5- iodouracil, 3-deoxyuridine, 6-thioguanine and 6- mercaptopurine, 5- or 6-azauridine, 5- or 6- azacytidine and 8-azaguanine which are incorporated into DNA prior to cell division.  Intercalating agents These are the molecules that can insert between bases in DNA base pairs, causing mutation during replication. Examples: Ethidiumbromide, Proflavine and Daunorubicin. Ethidiumbromide  Proflavine It also called Proflavin and Diaminoacridine , is an acriflavine derivative, a disinfectant bacteriostatic against many gram-positivebacteria. Daunorubucin is most commonly used to treat specific types of leukemia such as Acute myeloid leukemia , Acute lymphocytic leukemia) and also for the treatment of Neuroblastoma. Daunorubicin  DNA Polymerase Inhibitors Acyclovir Gancyclovir Guanosine Natuarlly occuring Inhibitors of Viral DNA Polymerase Nitorgen base essential in DNA Replication  Aphidicolin Inhibits DNA Polymerase-ε in Eukaryotes  DNA DAMAGE  DNA is easily damaged under normal physiological conditions.  The return of damaged DNA to its normal sequence and structure is called Repair.  Many different kinds of physical & chemical agents damage DNA. Some of these are:- 1) Endogenous agents 2) Exogenous agents Agents that damage DNA can be mutagenic, cytotoxic or both. DNA damaging agents that cause mutations are called Mutagens.  Types of DNA Damage The damages done to DNA by physical, chemical and environmental agents can be broadly classified into four categories with different types. Single Base Alterations Double Base Alterations  DNA damaging Agents Spontaneous Agents Highly reactive oxygen radicals produced as a by products during normal cellular respiration as well as by other biochemical pathways. Reactive Oxygen Species (ROS) : Hydrogen peroxide (H2O2) Hydroxyl radicals (OH.- ) – Most potent Superoxide (O2 ).- ✓ ROS causes DNA damage such as Oxidation of Nitrogen Bases, deoxy Ribose and Strand breaks. Radiation can cause mutations  Radiation  The high energy electromagnetic radiation to the exposure of which cell experience considerable damage to their DNA are: 1. Ultraviolet light:  The major type of damage caused by UV light is divided into three bands: I. UV-A (321-400 nm) II. UV-B (296-320 nm) III. UV-C (100-295 nm) 2. X- Rays 3. Gamma Rays  Through these direct damage takes place when DNA or water tightly bound to it absorbs the radiation. Indirect damage takes place when water or other molecules surrounding the DNA absorbs the radiation & form reactive species that then damage DNA. Effect of UV on DNA structure  Chemicals Agents 1) Deaminating Agents: Sodium Nitrite (NaNO2) Sodium Nitrate (NaNO3) Nitrosamine Nitrous Acid (HNO2) 2) Alkylating Agents: Dimethyl sulfate (DMS) Dimethyl nitrosamine Nitrogen mustard  Mutations Mutation refers to a change in the DNA structure of a gene. The substances (chemicals) which can induce mutations are collectively known as mutagens. The changes that occur in DNA on mutation are reflected in Replication, Transcription and Translation. Mutations occur in 2 ways: 1) Spontaneous mutations: Mistakes in DNA replication. 2) Induced mutation: Caused by Mutagens. 1) Point mutations : A point mutation or single base substitution, is a type of mutation that causes the replacement of single base nucleotides with another nucleotides of DNA. Substitutions (a) Transitions : In this case, a purine (or) a pyrimidine) is replaced by another. (b) Transversions : These are characterized by replacement of a purine by a pyrimidine or vice versa. Substitution  Point Mutations Silent Mutation : UCA UCU Serine Serine Missense Mutation : UCA ACA Serine Threonine Nonsense Mutation : UGG UGA Tryptophan Stop Codon UAU UA A Tyrosine Stop Codon UAC UA G Tyrosine Stop Codon  Missense mutation A point mutation can also change a codon so that a different protein is specified, a non synonymous change.. Sickle Cell Anemia 113 Normal red blood cell Sickle cell RBC 2). Frameshift mutations : These occur when one or more base pairs are inserted in or deleted from the DNA, respectively, causing insertion (or) deletion mutations. deletion DNA REPAIR MECHANISMS  Base Excision Repair  For correction of specific Chemical damage in DNA  Uracil  Hypoxanthine  3-methyl Adenine  Formamido pyrimidine  5,6 - Hydrated Thymine Base Excision Repair Deaminated (BER) Cytosine Variety of DNA glycosylases, for different types of damaged bases. AP endonuclease recognizes sites with a missing base; cleaves sugar- phosphate backbone. Deoxyribose phosphodiesterase removes the sugar-phosphate lacking the base. Nucleotide Excision Repair (NER)  Used by the cells to repair bulky DNA damages  Non specific DNA damage  Chemical adducts …  UV photoproducts Xeroderma pigmentosum (XP) is a rare autosomal recessive disease. The affected Patients are photosensitive and susceptible to Skin cancers. It is due to a defect in the Nucleotide Excision Repair of the damaged D NA. Mismatch repair Mismatch repair system is an excision/resynthesis system that can be divided into 4 phases: (i) recognition of a mismatch by MutS proteins (ii) recruitment of Repair enzymes (iii) excision of the incorrect sequence (iv) resynthesis by DNA polymerase using the parental strand as a template. Parental Srand DNA REPAIR DISORDERS Xeroderma Pigmentosum Transmitted as autosomal recessive disorder. Genetic defect: DNA repair mechanisms are defective. DNA damage produced by UV irradiation specially thymine dimers, cannot be incised. Results from inborn deficiency of the enzyme “nicking endonuclease”. Clinical Manifestations : Increased cutaneous sensitivity to UV rays of sunlight. Produces blisters on the skin. Dry keratosis, hyperpigmentation and atrophy of skin. May produce corneal ulcers. Ataxia telangiectasia : A familial disorder. Inheritence: Autosomal recessive Increased sensitivity to X-rays and UV rays is seen. Clinical manifestations : Progressive cerebellar ataxia. Oculocutaneous telangiectasia. Frequent sin pulmonary infections. Lymphoreticular neoplasms are common in this condition. IgE deficiency has been demonstrated in 67 per cent of cases. Bloom’s Syndrome Chromosomal breaks and rearrangements are seen in this condition. Genetic defect: Defective DNA-ligase. Clinical Manifestations – Facial erythema – Photosensitivity Fanconi’s Anaemia : An autosomal recessive anemia. Defective gene is located in chromosomes 20q and 9q. Defect: Defective repair of cross-linking damage. Characterized by An increased frequency of cancer and by chromosomal instability.  Hereditary Nonpolyposis Colon Cancer (HNPCC) Most common inherited cancer. Defect: Faulty mismatch repair. Genetic defect has been located in chromosome 2, The located gene is called hMSH-2. Mutations of hMSH-2 account for 50 to 60 per cent of HNPCC cases.  Recombination Genetic diversity in a species is maintained through both mutation and recombination. Mutation alters single genes or small groups of genes in an individual, whereas recombination redistributes the contents of a genome among various individuals during reproduction.  Recombination basically involves the exchange of genetic information. There are mainly two types of recombination. - Homologous Recombination (Meiosis). - Transposition. Recombination is mediated by the breakage and joining of DNA strands.  Recombination ABCDEFGHIJKLMNOPQRSTUVWXYZ abcdefghijklmnopqrstuvwxyz Exchange of genes between the chromatids of Chromosomes ABCDEFGhijklmnoPQRSTUVWXYZ abcdefgHIJKLMNOpqrstuvwxyz  Homologous Recombination In eukaryotes, homologous genetic recombination can have several roles in replication and cell division, including the repair of stalled replication forks. Recombination occurs with the highest frequency during meiosis, the process by which diploid germ-line cells with two sets of chromosomes divide to produce haploid gametes— sperm cells or ova in higher eukaryotes—each gamete having only one member of each chromosome pair. Holliday Junction Model for Homologous Recombination  Transposition Transposition primarily involves the movement of specific pieces of DNA in the genome. The mobile segments of DNA are called transposons (or) transposable elements. Types of Transposition : Two types 1). DNA transposition 2).Retrotransposition DNA transposition : Some transposons are capable of direct transposition of DNA to DNA. This may occur either by replicative transposition or conservative transposition. DNA transposition is less common than retro transposition in case of eukaryotes. However, in case of prokaryotes, DNA transposons are more important than RNA transposons.  Retrotransposition Transposition involving RNA intermediate represents Retrotransposition. A copy of RNA formed from a transposon( also called as retro transposon). Then by the enzyme Reverse transcriptase, DNA is copied from the RNA. The newly formed DNA which is a copy of the transposon gets integrated into the genome. This integration may occur randomly on the same chromosome or/ on a different chromosome.