DNA Replication.pdf

Unit 2: Replication of DNA |1 DNA Replication: The biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication is significant because after cell division, each new daughter cell should contain a...

Unit 2: Replication of DNA |1 DNA Replication: The biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication is significant because after cell division, each new daughter cell should contain a copy of the full amount of DNA material. DNA replication enables the flow of genetic information from one generation to the next. Modes of DNA Replication: Conservative Replication Semi-Conservative Replication Dispersive Replication After being used as Each of the 2 parental DNA strands act After replication, two templates the 2 parental as a template for new DNA strands to daughter DNAs have DNA strands re-base pair be synthesized, but after replication, alternating segments of with each other and the each parental DNA strand base pairs parental DNA and newly- two newly synthesized with the complementary newly- synthesized DNA daughter strands, also synthesized strand. interspersed on both base pair with each other. strands. One of the 2 DNA Both DNA molecules contain one molecules after replication parental or “old” strand and one would be “all-old” and the daughter or “new” strand. other would be “all new”. Proof for semiconservative replication: Experiment performed in 1958 by two researchers: Matthew Meselson and Franklin Stahl. Meselson – Stahl Experiment: 1. E. coli cells were grown for several generations in a medium with 15N –NH4Cl (15N=“heavy” isotope of Nitrogen). DNA of these cells had a higher density than cells grown in normal 14N medium. 2. E. coli cells with only 15N in their DNA were then transferred to a 14N medium and were allowed to divide and grow for several generations. DNA was extracted periodically and was compared to pure 14N DNA and 15N DNA, using Cesium chloride density gradient centrifugation. Results: 1. After 1 generation time (F1) on 14N-NH4Cl, the DNA had intermediate density (between 14N and 15N); showing a single band in the density gradient between light and heavy DNA, indicating semi-conservative replication, with 1 strand of 15N DNA, and 1 of 14N DNA. 2. After 2 generations on 14N-NH4Cl, DNA showed 2 bands, one similar to normal light DNA and the other similar to F1 hybrid DNA.  Unit 2: Replication of DNA |2 Conservative replication would have yielded two DNA bands for generation 1 (i.e., F1) cells. Therefore, DNA replication is not conservative. Dispersive replication would have produced a single band for each generation and the band would have been found at successively lighter density positions in the gradient. Thus, the Meselson-Stahl experiment conclusively proved that DNA replication is semi-conservative. Key features of DNA Replication 1. DNA replication is a semi-conservative process (notes in table) 2. DNA replication is bi-directional Replication starts at a specific site called the origin of replication (ori), and the double strands in DNA separate forming a replication bubble. Thus, replication proceeds in both directions from the origin. 3. DNA replication requires a primer A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis. DNA polymerases, cannot initiate de novo synthesis of a DNA. Therefore, it requires a free 3'-OH end of a nucleotide to attach more nucleotides. So, 3'-OH end is provided by synthesizing a primer on both template strands. 4. DNA replication direction is from 5' to 3' During synthesis of daughter DNA strands, nucleotides are added at the 3' end of the growing strand. Therefore, replication direction is said to be 5' to 3'. 5. DNA replication is a semi-discontinuous process Leading strand is replicated continuously. Lagging strand is replicated discontinuously. Okazaki fragments- short fragments of DNA due to synthesis of the lagging strand. Enzymes involved in DNA replication 1. DNA Polymerases DNA Polymerases are the enzymes that catalyze synthesis of DNA by the formation of a phosphodiester bond between the nucleotides. DNA polymerases cannot initiate de novo synthesis of a DNA chain. Primer addition allows DNA polymerases to add nucleotides and synthesize daughter DNA. In E. coli, 5 different types of DNA polymerases are found; DNA polymerase I, II, Ill, IV, V.  Unit 2: Replication of DNA |3 DNA Polymerase I DNA Polymerase II DNA Polymerase III Main DNA replication, DNA repair Major enzyme for DNA function recombination and repair (backup of DNA pol III) replication 1. 5’→3’ polymerase 5’→3’ polymerase 5’→3’ polymerase 2. 5’→3’ exonuclease (Primer - -------- - ----- - removal from lagging strand) Activity 3. 3’→5’ exonuclease activity 3’→5’ exonuclease activity 3’→5’ exonuclease (proofreading). (proofreading). (proofreading). 4. Gap filling single polypeptide (103Da) 7 subunit protein (90Da) complex of 10 proteins, arranged Structure as 4 subcomplexes (830Da) Coded by coded by polA gene. coded by polB gene. Coded by polC gene polymerization and (1500/association) polymerization and processivity Features processivity rate is low rate is maximum. (200/association) (>500,000/association) DNA Polymerase IV is coded by dinB gene. Its main role is in DNA repair during SOS response, when DNA replication is stalled at the replication fork. DNA Polymerase V is also involved in translesion synthesis during SOS response and DNA repair. It is made up of UmuC monomer and UmuD dimer. DNA polymerase III: Main enzyme Subcomplex 1: Catalytic core Made up of 2 copies of the catalytic cor. Each catalytic core consists of: α subunit (polymerase activity), ε subunit (proofreading activity) and θ subunit (exonuclease activity). Subcomplex 2: Sliding Clamp Sliding clamp is made up of β subunit. It has a ring-shaped structure. It holds the catalytic cores on template strands. Two copies of beta clamp are present. Subcomplex 3: Dimerization component It is made up of τ subunit. It links catalytic cores together. Two copies of dimerization component are present. Subcomplex 4: Clamp Loader This subcomplex is made up of a group of 5 proteins γ, δ, δ’, χ and Ψ. DNA polymerase I 1st DNA polymerase enzyme discovered in E. coli. Second DNA polymerase which plays a crucial role in DNA replication. It has low processivity and polymerization rate. Therefore, it is not the main enzyme that catalyzes synthesis of DNA. Known as the Kornberg enzyme. (Characteristics in table)  Unit 2: Replication of DNA |4 2. Primase (Dna G) : − Primase is a type of RNA polymerase. − Carries out de novo synthesis of ribonucleotide chain. It synthesizes a short stretch (11-12 bp) of RNA nucleotides or primers, with free 3'-OH end for DNA polymerases to function. − Primase enzyme is a single polypeptide of 60 kD. 3. DNA Gyrase : − DNA strands open and unwind in the replication bubble ahead of replication fork. This causes supercoiling and torsional strain in the DNA. − DNA Gyrase relieves the torsional strain created due to supercoiling. It makes temporary nicks in the helix to release the tension, then seals the nicks to avoid permanent damage. − DNA gyrase is a type II topoisomerase enzyme. It creates nicks in double-stranded DNA. − It is a tetramer with two subunits, GyrA and GyrB. GyrA nicks and seals the DNA strands and GyrB provides energy by ATP hydrolysis. 4. DNA Ligase : − DNA ligase catalyzes the formation of a phosphodiester bond at the end of replication process between adjacent 3'-OH and 5'-phosphate groups in a DNA molecule. − It uses NAD+ as a cofactor. Ligase forms a complex with AMP. − It attaches to the 5'phosphate termini of the nick, and forms a phosphodiester bond with 3'-OH termini. Regulatory proteins in prokaryotic DNA replication 1. DnaA − DnaA protein is the first protein that binds at the site of origin. Therefore, it is called as an initiator protein. − Its binds at 9-mer sequence repeats present in origin of the replication site. Dna A uses ATP. − Binding of Dna A protein catalyzes the opening of the double helix at 13-mer repeats. 2. Helicase: Dna B − DnaB is a helicase enzyme that unwinds the DNA double helix. − It is a hexamer and ring-shaped. It unwinds the double-stranded DNA by breaking hydrogen bonds. − It uses ATP as an energy source. 3. Helicase loader: Dna C It binds to single-stranded DNA and loads DnaB. It opens the DnaB ring and loads it on DNA strands. After DnaB is loaded, it separates from the complex. 4. Single-Stranded Binding (SSB) Proteins SSB proteins bind to single strands of DNA in the replication fork. Binding is sequenceindependent. By doing this they keep the separated strands from reannealing. SSB protein is a tetramer.  Unit 2: Replication of DNA |5 Process of DNA replication (elongation phase diag.) The process of DNA replication is divided into 3 steps: Initiation, Elongation and Termination. 1. Initiation DNA replication starts at a specific point on DNA strands, called Origin of replication ‘OriC’. OriC contains 245 bp long sequence of DNA with 2 repeat motif sequences; 9 mer and 13 mer. Five copies of 9mer (5’TTATCCACA3’) and 3 copies of 13 mer (5’GATCTATTTATTT3') sequence repeats are present in OriC. Steps: 1. Initiator protein DnaA, binds to 9-mer repeats in OriC and facilitates strand separation at 13-mer repeats and formation of an open complex. 2. DnaC protein (Helicase loader) catalyzes the opening of DnaB ring (Helicase) and loads DnaB around the lagging DNA strands. DnaB unwinds and separates the duplex strands by using ATP. 3. Separated strands are inhibited from reannealing by binding Single-Stranded Binding proteins in a sequence- independent manner. 4. DnaB protein recruits DnaG protein (DNA Primase) on template strands to synthesize short RNA primers. Primers provide a free 3'-OH end for DNA polymerase III to initiate DNA replication. 5. After primer synthesis, DnaC separates from DnaB. The open complex of regulatory proteins, DNA strands, and primer is called as a Primosome. 2. Elongation 1. DNA Polymerase III catalyses the addition of dNTPs on the 3’-OH end terminus of primers by the formation of phosphodiester bonds. Template strand directs which of the 4 dNTPs are added. Mg+2 and Zn+2 ions bound to DNA Polymerase III help in dNTP addition.  Unit 2: Replication of DNA |6 2. In the 2 replication forks, leading strands are synthesized continuously from a single primer in 5' to 3' direction by DNA polymerase III enzyme. 3. Lagging strands are synthesized discontinuously using multiple primer in 5’ to 3’ direction by DNA Polymerase III enzyme. These DNA fragments synthesized on lagging strand are called Okazaki Fragments. 4. DNA polymerase I follows DNA polymerase III, proofreads, removes primers and fills the gap created due to the removal of primers. 5. DNA ligase forms a phosphodiester bond between 5’-phosphate of first nucleotide added by DNA polymerase III and 3’-OH terminus of last nucleotide added by DNA polymerase I. 3. Termination 1. Termination of replication occurs when the replication fork reaches the Ter sequence/region on DNA strands. 2. Terminus region is called Ter Sequence which is a 23 bp long sequence. 3. Tus (Terminus Utilization Substance) protein binds to Ter Sequence forming a Ter-Tus complex. 4. Ter -Tus complex blocks the passage of helicase enzyme and replication terminates. Inhibitors of replication Quinolene antibiotics work on the subunits of gyrase (topoisomerase) enzymes which controls the supercoiling of DNA and reduces torsional strain. GyrA cuts and rejoins DNA strands. GyrB provides energy by ATP hydrolysis. Quinolones bind to gyrase-DNA complex and stabilize it leading to cleavage of DNA across both strands 1. Nalidixic Acid, Norfloxacin, and Ciprofloxacin bind to GyrA subnunit. 2. Novobiocin binds to GyrB protein. Models of Replication in Prokaryotes & Eukaryotes: Rolling Circle/Sigma and Theta 1. Sigma Model (Rolling circle replication) Rolling circle replication is a mechanism used by some plasmids and viruses. Replication starts by nicking and unrolling one strand. For a single-stranded genome, this is preceded by using the still-circular strand as a template for DNA synthesis; for a doublestranded genome, the unrolled strand is used as a template for DNA synthesis. Rolling circle replication is unidirectional. Steps involved in sigma model of replication: 1. One strand of double stranded DNA is nicked and rolls out (therefore it is named rolling circle). The one strand that rolls, acts as a template for the synthesis of new strand. 2. Nick occurs at the origin of replication by a sequence-specific endonuclease enzyme producing a free 3'-OH (hydroxyl) and 5'-PO4 (phosphate) ends. 3. 3’-hydroxyl (OH) end is extended by replication enzymes and lengthened. 4. The growing point continues around the circular DNA template. 5. 5’ end of the strand is displaced to form an ever-lengthening tail. 6. A single-stranded tail, often composed of more than one genome copy, is generated can be converted to the double-stranded form by synthesis of a complementary strand.  Unit 2: Replication of DNA |7 2. Theta Model In circular DNA, bidirectional replication from an origin forms an intermediate structure resembling the Greek letter theta (θ). Steps involved in theta model of replication: 1. The replication of chromosomal DNA begins at origin of replication. 2. Synthesis of DNA occurs at the replication fork, the place at which the DNA helix is unwound and each strand is replicated. 3. Two replication forks move outward from the origin until they have copied the whole replicon (genome that contains an origin that is replicated as a unit). 4. When the replication forks move around the circular chromosomes, a structure is formed which is shaped like the Greek letter theta (θ). 5. Because the bacterial chromosome is a single replicon, the forks meet on the other side and two separate chromosomes are released. Eukaryotic DNA Replication DNA Replication in eukaryotes shares similarities with the process and enzymes in prokaryotes. As with prokaryotes, DNA replication in eukaryotes occurs in three stages: initiation, elongation, and termination, which are aided by several enzymes. During initiation, proteins bind to the origin of replication while helicase unwinds the DNA helix and two replication forks are formed at the origin of replication. During elongation, a primer sequence is added with complementary RNA nucleotides, which are then replaced by DNA nucleotides. During elongation DNA polymerase synthesizes the leading strand in 5’ to 3’ direction continuously, while the lagging strand is synthesized discontinuously i.e. in pieces, called Okazaki fragments. During termination, RNA primers are removed and replaced with new DNA nucleotides and the backbone is sealed by DNA ligase.  Unit 2: Replication of DNA |8 Eukaryotic Chromosomes have multiple Origins. The process of replication of eukaryotic DNA is more complex due to: Large linear chromosomes, multiple origins of replication per chromosome and tight packaging of DNA around histone proteins. The DNA Replication rate is thus lower in eukaryotes. Eukaryotic DNA is bound to proteins known as histones to form nucleosomes. During initiation, the DNA is made accessible to the proteins and enzymes involved in the replication process. There are specific chromosomal locations called origins of replication where replication begins. Eukaryotic chromosomes have multiple origins of replication. Considering the size of eukaryotic chromosomes, this is necessary to increase the rate of replication. Each of these origins defines a replicon, or the stretch of the DNA that is replicated from a particular origin. Linear Model of Replication in Eukaryotes 1. To replicate eukaryotic DNA in short time, there are many replicons (1 replication origin every 10-100μm along DNA). 2. Replication begins at the origin, forms 2 replication forks (bidirectional replication) that move in opposite directions; forming a “replication bubble”. 3. Replication bubbles ‘grow’ in size and adjacent bubbles fuse. 4. Parent linear double stranded DNA molecule is copied semi-conservatively to form 2 linear double helical daughter DNAs.  Unit 2: Replication of DNA |9 Eukaryotic DNA Polymerase Like prokaryotic cells, eukaryotic cells also have many DNA polymerases, which perform different functions, The nuclear DNA replication is mainly done by DNA polymerase 𝝳 and 𝜶. There are at least 15 DNA polymerases identified in human beings. DNA polymerase 𝝳 –main enzyme for replication in eukaryotes. It also has 3’→5’ exonuclease activity for proofreading. DNA polymerase 𝜶 – The main function of DNA polymerase 𝜶 is to synthesize primers. The smaller subunit has a primase activity. The largest subunit has polymerization activity. It forms a primer for Okazaki fragments, which is then extended by DNA polymerase 𝝳. DNA polymerase 𝟄 – The main function is DNA repair. It removes primers for Okazaki fragments from the lagging strand. DNA polymerase 𝝲 – It is the main replicative enzyme for mitochondrial DNA. Differences between prokaryotic DNA and eukaryotic DNA Replication Initiation Prokaryotic Eukaryotic Site Cytoplasm Nucleus Phase C phase of cell cycle S phase of cell cycle Speed Fast Slow Origin Single Multiple Initiator protein Dna A (E. coli) Origin Recognition Protein C 1-6 (Yeast) Helicase loader Dna C Cdc 6, Cdt 1 Helicase Dna B MCM 2-7 Stabilization of strands Single stranded binding RPA (Replication protein A) proteins Primase Dna G DNA polymerase α Replication Elongation DNA Polymerases 5 types 15 types Replication enzymes DNA Polymerase III DNA Polymerase δ – Lagging strand DNA Polymerase ε – Leading strand Primer removal DNA Polymerase I RNAse H/ Flap endonuclease I Gap filling DNA Polymerase I DNA Polymerase δ – Lagging strand DNA Polymerase ε – Leading strand Replication Termination Ter sequence & Tus Joining of 2 replication forks protein Telomerase also aids in termination