DNA Replication Notes PDF

DNA replication  By the end of this session you will be able to: describe the process of DNA replication (both strands) describe the function of different enzymes used in DNA replication describe the direction of DNA polymerisation and the reason why describe error correction...

DNA replication  By the end of this session you will be able to: describe the process of DNA replication (both strands) describe the function of different enzymes used in DNA replication describe the direction of DNA polymerisation and the reason why describe error correction in DNA replication describe DNA mutation and consequence of DNA mutation  DNA: Molecule of Heredity For a cell to produce two genetically identical daughter cells, first DNA replication must occur. Maintaining this genetic identity requires the continued surveillance and repair of DNA. Immediate cell survival depends on preventing changes in its DNA. BUT long-term survival of species and evolution requires DNA be changeable over generations.  The bacterial cell cycle 1. the period between division (birth) and the initiation of chromosome replication (the B period); 2. the period required for chromosome replication (the C period); 3. the time between the completion of chromosome replication and the completion of cell division (the D period). The bacterial cells (in this case, Escherichia coli) are outlined in black and contain highly schematic chromosomes (purple ovals) with oriC regions shown as green circles. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2887316/  Base-Pairing Underlies DNA Replication and DNA Repair From Watson, JD and Crick FHC, 1953, Nature 171:964- 967 'it has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material….' '..where each of the complementary strands acts as a template for the synthesis of a new strand complementary to it.’  Base-Pairing Underlies DNA Replication and DNA Repair DNA replication requires a template DNA templating is where a sequence of a DNA strand (or selected portions of) is copied by complementary base- pairing (A with T; G with C) into the new complementary DNA sequence. Requires recognition of each nucleotide in DNA template strand by a free (unpolymerized) complementary nucleotide. Requires that the two strands of DNA helix be separated.  Base-pairing Pattern of base-paring is complementary A forms two hydrogen bonds with T G forms three hydrogen bonds with C Two strands of single DNA molecule are not identical, each strand specifies the other by base-pair complementarity © McGraw Hill, LLC 9  DNA: Anti-parallel and complementary A+T 2 H-bonds Polarity of strand Purine-pyrimidine based on direction of pairs sugar-phosphate backbone G+C 3’ end is OH 5’ end is PO4 3 H-bonds The two strands of a single DNA molecule have opposite polarity to one another Strands are referred as having either 5′-to-3′ or 3′-to-5′ polarity  Nucleotide Structure Figure 3.14 Access the text alternative for slide images. © McGraw Hill, LLC 11  The polynucleotide strands held together by H bonding between base pairs A+T 2 H-bonds Purine-pyrimidine pairs G+C 3 H-bonds  DNA Replication: Requirements are… 1) Something to copy  Parental DNA molecule (template) 2) Something to do the copying  Specific Enzymes, Primer (short nucleic acid sequence as starting point for DNA polymerisation) 3) Building blocks to make copy  Nucleotide triphosphates of all the different nucleotides must be present. So dATP, dGTP, dCTP and dTTP 4) Somewhere to start  Origin of replication on the original DNA 13  Three Possible Models of DNA Replication 1 1. Conservative model – both strands of parental DNA remain intact; new DNA copies consist of all new molecules 2. Semiconservative model – daughter strands each consist of one parental strand and one new strand 3. Dispersive model – new DNA is dispersed throughout each strand of both daughter molecules after replication © McGraw Hill, LLC 14  Three Possible Models of DNA Replication 2 Access the text alternative for slide images. © McGraw Hill, LLC 15  Meselson and Stahl – 1958 Bacterial cells were grown in a heavy isotope of nitrogen, 15 N After several generations, the DNA of these bacteria was denser than normal DNA Cells were switched to media containing lighter 14 N DNA was extracted from the cells at various time intervals and centrifuged to separate out by weight © McGraw Hill, LLC 16  The Meselson–Stahl experiment Meselson, M., Stahl, F., (1958) “The replication of DNA in Escherichia coli,” PNAS, 44(7):671-682, Fig. 4a Thus, DNA replication is semi-conservative. © McGraw Hill, LLC 17 © McGraw Hill, LLC  DNA replication is semi-conservative © McGraw Hill, LLC  Stages of DNA replication Initiation – replication begins Elongation – new strands of DNA are synthesized by DNA polymerase Termination – replication is terminated Prokaryotes and eukaryotes have similar process Eukaryotes have greater complexity than prokaryotes © McGraw Hill, LLC 20 21 © McGraw Hill, LLC  Prokaryotic Replication E. coli used as model system for understanding universal attributes of replication Single circular molecule of DNA Replication begins at the origin of replication Proceeds in both directions around the chromosome Replicon – DNA controlled by an origin © McGraw Hill, LLC 22  Prokaryotic DNA Replication E. coli used as model system for understanding universal attributes of replication Single circular chromosome of DNA Replication begins at the origin of replication Proceeds in both directions around the chromosome Many different enzymes involved © McGraw Hill, LLC 23 Origin of replication specific sequence Double-stranded 5’ 3’ DNA 3’ 5’ Double helix opened with aid of initiator proteins Single-stranded DNA ready for DNA synthesis 2 Replication forks 5’ 3’ 3’ 5’ © McGraw Hill, LLC Parental DNA = template  DNA replication requires an Origin of replication (in prokaryotes OriC) DNA replication initiates with the loading of the ring- shaped helicase at the Replication origin, OriC. The initiator protein DnaA binds to specific sequences within the OriC region and then mediates DNA unwinding and supports the recruitment of the enzyme helicase and its loader to the bacterial chromosome. © McGraw Hill, LLC DNA Replication in Prokaryotes: STEP 1: Initiation Origin of replication is called OriC (specific nucleotide sequence). DnaA = promotes unwinding and strand separation of DNA by binding to specific sites on DNA HU = is a histone-like protein, which promotes negative supercoiling , makes compact DNA accessible for replication DnaB= is a helicase enzyme that continues to unwind the DNA, forming the replication fork DnaC= associated protein that helps ‘open up’ the helicase enzyme to help it bind to DNA © McGraw Hill, LLC  The polynucleotide strands held together by H bonding between base pairs A+T 2 H-bonds Purine-pyrimidine pairs G+C 3 H-bonds © McGraw Hill, LLC  DNA Replication in Prokaryotes: Elongation New strand Template strand 5′ end 3′ end 5′ end 3′ end Sugar A T A T Phosphate Base C G C G G C G C DNA polymerase 3′ end A T A C Pyrophosphate 3′ end C Nucleoside triphosphate 5′ end 5′ end © McGraw Hill, LLC  Nucleotides joined by together by condensation reaction. Hydroxyl group (OH) group on carbon 3 (3’) of ribose of one nucleotide and phosphate group on carbon 5 (5’) of ribose group of another nucleotide. REMEMBER about 3’ and 5’ orientation of DNA strands © McGraw Hill, LLC © McGraw Hill, LLC  DNA Elongation Enzymes called DNA polymerases catalyze the elongation of new DNA. Most DNA polymerases require a primer and a DNA template strand. 3 types of Polymerase enzyme: Pol I, Pol II, Pol III DNA polymerase III (Pol III) is main replication polymerase Rate of elongation is about 500-1000 nucleotides per second in bacteria and 50 per second in human cells. Nucleotides are linked by a condensation reaction forming ester bond. (see lecture 11) Nucleotides can only be added to 3’ end of DNA strand. Elongation and Pol III need a starting primer  DNA Polymerases DNA polymerase I (Pol I) DNA polymerase I, II & IIII Acts on lagging strand to remove primers and replace with DNA (5’-to-3’exonuclease activity) DNA polymerase II (Pol II) Involved in DNA repair processes DNA polymerase III (Pol III) Main replication enzyme All 3 have 3′-to-5′ exonuclease activity – proofreading DNA polymerase I only Exonuclease enzyme –is one which degrades nucleic acids from an end © McGraw Hill, LLC  DNA Polymerases Summary DNA Polymerase I – Three different activities Template directed 5’-3’ polymerase Proof reading (3’-5’ exonuclease) Error correcting (5’-3’ exonuclease) DNA Polymerase II and III Template directed 5’-3’ polymerase Proof reading (3’-5’ exonuclease activity) Many other proteins involved DNA polymerase III (Pol III) Main replication enzyme © McGraw Hill, LLC  Requirement for a primer DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3′ end The initial nucleotide strand is a short RNA primer An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template The primer is short (5–10 nucleotides long), and the 3′ end serves as the starting point for the new DNA strand © McGraw Hill, LLC The sliding clamp is a ring-shaped protein that encircles duplex DNA, binds to the DNA Origin of replication polymerase and tethers it to the DNA template, preventing its dissociation and providing high processivity. 3′ 5′ 5′ RNA primer 3′ “Sliding clamp” 5′ DNA pol III Parental DNA DNA sliding clamps can be 3′ used as therapeutic targets 5′ Helicases – use energy 5′ from ATP to unwind DNA 3′ Single-strand-binding proteins (SSBs) coat 5′ strands to keep them apart © McGraw Hill, LLC Replication fork is partial Origin of replication opening of helix formed where double stranded DNA is being unwound 3′ 5′ 5′ RNA primer 3′ “Sliding clamp” 5′ DNA pol III Parental DNA 3′ 5′ 5′ 3′ 5′ © McGraw Hill, LLC © McGraw Hill, LLC  Antiparallel Elongation The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication DNA polymerases add nucleotides only to the free 3′ end of a growing strand; therefore, a new DNA strand can elongate only in the 5′ to 3′ direction Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork  How, then, is overall 3’-to-5’ DNA chain growth achieved? In the late 1960s molecular biology researchers Reiji and Tsuneko Okazaki added highly radioactive 3H-thymidine to dividing bacteria for a few seconds, so that only the most recently replicated DNA became radiolabeled. This experiment revealed the transient existence of pieces of DNA that were 1000–2000 nucleotides long, now commonly known as Okazaki fragments, at the growing replication fork Reiji and Tsuneko Okazaki, Nagoya University, Japan  Elongation – The lagging strand To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork The lagging strand is synthesized as a series of segments called Okazaki fragments Okazaki fragment - RNA primer and short piece of DNA Once the RNA primer is removed and replaced with DNA by DNA polymerase I the sections are joined together by DNA ligase Fig. 16-15 Overview Origin of replication Leading strand Lagging strand Primer Lagging strand Leading strand Overall directions of replication Origin of replication 3′ 5′ 5′ RNA primer “Sliding clamp” 3′ 5′ DNA poll III Parental DNA 3′ 5′ 5′ 3′ 5′ Fig. 16-17 Overview Leading strand Origin of replicationLagging strand Single- strand binding protein Lagging strand Leading strand Overall directions of replication Helicase Leading strand 5′ DNA pol III 3′ Primer Primase 3′ 5′ Parental DNA 3′ 5′ DNA pol III Lagging strand DNA ligase 3′ 5′ DNA pol I 4 3′ 3 2 1 5′ Overall direction of replication  Elongation - Lagging-strand Elongation of the lagging strand is discontinuous synthesis and requires multiple enzymes: DNA Pol III (like leading strand) Primase - Makes RNA primer for each Okazaki fragment DNA Pol I - Removes all RNA primers and replaces with DNA DNA ligase – joins Okazaki fragments to form complete strands Termination occurs at specific site: DNA gyrase unlinks two copies 45  Replication is semi discontinuous Leading strand synthesized continuously from an initial primer Lagging strand synthesized discontinuously with multiple priming events DNA fragments on the lagging strand are called Okazaki fragments, must be connected together 47  Overview Origin of replication Leading strand Lagging strand Primer Lagging strand Leading strand Overall directions of replication  Unwinding the helix causes torsional strain Unwinding of DNA introduces torsional strain in the molecule that can lead to additional twisting of the helix, called supercoiling Topoisomerases are enzymes that prevent supercoiling DNA gyrase is the topoisomerase involved in DNA replication that relieves the torsional strain © McGraw Hill, LLC 49  Replication is bidirectional from a unique origin Termination of DNA replication occurs when two replication forks meet. This can happen at specific termination sites on the DNA molecule, during which the following events occur, although not necessarily in this order: Forks converge until all intervening DNA is unwound Any remaining gaps are filled and ligated and RNA primers are removed Dissembly of replication proteins which are unloaded. © McGraw Hill, LLC 50 An additional range of ENZYMES are then required for DNA replication  Replication in eukaryotes Follows similar principles, but the enzymes are more numerous and complex Rate of replication is slower for eukaryotes than prokaryotes The prokaryotic bacterium E. coli, replicates at a rate of approx. 1,000 nucleotides per second. In comparison, eukaryotic human DNA replicates at a rate of 50 nucleotides per second.  In eukaryotes histones/nucleosomes slow down the process as they physically impede the progress of the polymerases but………….  In eukaryotes there are many origins of replication rather than one – these are initiated more or less simultaneously https://www.embopress.org/doi/full/10.15252/emmm.201505965  Eukaryote Linear chromosomes have specialized ends Telomeres Specialized structures found on the ends of eukaryotic chromosomes Composed of specific repeat sequences Protect ends of chromosomes from nucleases Maintain the integrity of linear chromosomes Not made by replication complex Telomerase is an enzyme that synthesizes the telomere repeat sequences at the ends of strand Uses an internal RNA template (not the DNA itself) © McGraw Hill, LLC 55  Error correction mechanisms during replication – DNA polymerase Nucleotide affinity. The correct nucleotide has a higher affinity for the moving polymerase than does the incorrect nucleotide, because only the correct nucleotide can correctly base-pair with the template. The DNA polymerase can “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. This is because before the nucleotide is covalently added to the growing chain, the enzyme must undergo a conformational change. An incorrectly bound nucleotide is more likely to dissociate during this step than the correct one. Exonucleolytic proofreading (after addition)  DNA Replication with a Proofreading Polymerase Extension proceeds along the template strand at the 3' end of the newly synthesized strand. When the polymerase recognizes an error, the mismatched base is transferred to the exonuclease active site and the base is excised. The extended strand returns to the polymerase domain, re-anneals to the template strand, and replication continues. https://international.neb.com/tools-and-resources/feature-articles/polymerase-fidelity- what-is-it-and-what-does-it-mean-for-your-pcr Exonucleolytic proofreading: How does the enzyme sense whether a newly added base is correct? 1. An incorrect base will not pair correctly with the template strand. There is greater structural fluctuation, due to the weaker hydrogen bonding, and this will usually bring the strand to the the exonuclease site on the DNA polymease 2. Second, after the addition of a new nucleotide, the DNA translocates by one base pair into the enzyme. The newly formed base pair must be of the proper dimensions to fit into a tight binding site and participate in hydrogen-bonding interactions in the minor groove similar to those in the polymerization site itself.  Mismatch repair (Post replication, before methylation) Removal and replacement of mis-paired bases (not corrected during proofreading). Newly synthesised strands are recognised by the absence of methyl groups on GATC sequences  Mismatch repair enzymes Scans newly made DNA to see if there are any mispaired bases (e.g., a G paired to a T) Finds mismatches, cuts out the region of the mismatch. (The mismatched strand is recognized by the fact that it is not methylated). The gap created by the excision of the mismatch is then filled in correctly. In humans, there are 2 sets of mismatch enzymes Muro et al., (2015) DNA mismatch repair enzymes: Genetic defects and autoimmunity https://www.sciencedirect.com/science/article/pii/S0009898115000285?via=ihub  Mutation If the DNA-maintenance processes fails, resulting in permanent change in the DNA. Such a change is called a mutation. This can be fatal to an organism if in a vital position in the DNA sequence. Mutation rates are generally extremely low  The most common source of DNA mutation is error during replication There is an average mistake of 1 base pair every 10,000 Due to proofreading and repair mechanisms this rate declines to 1 every 1,000,000,000 Inherent in meiosis are assortment and cross-over events that lead to highly significant changes in germ line DNA sequences  Mutation rates are extremely low but are an essential component of evolutionary change Somatic mutations may or may not affect the individual but cannot affect the population Figure 5-1 Molecular Biology of the Cell (© Garland Science 2008) Background on DNA Mutations a. Mutation rates are extremely low but are an essential component of evolutionary change b. The most common source of DNA mutation is error during replication c. Environmental damage to the DNA is independent of DNA mutation but can also be the underlying cause  Environmental damage to the DNA is independent of DNA mutation but can also be the underlying cause 1. DNA damage is simply a chemical alteration to DNA, whereas DNA mutation is a change in one or more base pairs 2. DNA damage becomes DNA mutation when DNA replication proceeds without repairing the damage or by means of error-prone DNA repair systems  Common Types and Mechanisms of DNA Damage and Mutation a. The alteration of a single base pair (point mutation) can result from chemical damage followed by copying error b. The insertion or deletion of a single base pair (point mutation) during DNA replication c. Single-stranded and double-stranded breaks can result from electrophilic attack from reactive oxygen species  Summary DNA is the molecule of heredity The base pairing structure allows for DNA replication and repair DNA replication is a semi-conservative process, requiring a template and is bi-directional with leading and lagging strands Proof reading mechanisms ensure mutation rates are low However, mutation can occur and is an essential component of evolutionary change  DNA Replication in Prokaryotes 1. DNA unwinds at the origin of replication. 2. Helicase opens up the DNA-forming replication forks; these are extended bidirectionally. 3. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA. 4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling. 5. Primase synthesizes RNA primers complementary to the DNA strand. 6. DNA polymerase III starts adding nucleotides to the 3'-OH end of the primer. 7. Elongation of both the lagging and the leading strand continues. Lagging strand elongating by Okazaki fragments 8. RNA primers are removed by exonuclease activity of DNA polymerase I 9. Gaps are filled by DNA pol I by adding dNTPs. 10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds. https://www.youtube.com/watch?v=0Ha9nppnwOc