DNA Replication Lecture Notes PDF

The Central Dogma of Molecular Biology Biology 2101 FG Ayson – Sep 2024  central dogma of molecular biology - 2 The central dogma states that the information that occurs in our cells is: from existing DNA to make new DNA (...

The Central Dogma of Molecular Biology Biology 2101 FG Ayson – Sep 2024  central dogma of molecular biology - 2 The central dogma states that the information that occurs in our cells is: from existing DNA to make new DNA (DNA replication), from DNA to make new RNA (transcription), and from RNA to make new proteins (translation). Biology 2101 FG Ayson – Sep 2024  central dogma of molecular biology - 3 Genetic information flows only in one direction, from DNA, to RNA, to protein. Biology 2101 FG Ayson – Sep 2024  Central Dogma Summary of the flow of genetic information from DNA to proteins in eukaryotic cells. The DNA is transcribed into RNA in the nucleus. RNA is translated into protein in the cytoplasm. Prokaryotic cells do not have a nucleus; transcription and translation proceed in their cytoplasm. Biology 2101 FG Ayson – Sep 2024  DNA Replication Replication is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules. DNA replication is one of the most basic processes that occurs within a cell. Each time a cell divides, the two resulting daughter cells must contain exactly the same genetic information, or DNA, as the parent cell. To accomplish this, each strand of existing DNA acts as a template for replication. There are almost three billion base pairs of DNA to be copied. Replication uses DNA polymerases which are molecules specifically dedicated to just copying DNA. Biology 2101 FG Ayson – Sep 2024  DNA replication - 2 Biology 2101 FG Ayson – Sep 2024  The Cell Cycle During the G1, S, and G2 phases, the cell grows continuously. During M phase, growth stops, the nucleus divides, and the cell divides in two. DNA replication is confined to the part of interphase known as S phase. G1 is the gap between M phase and S phase; G2 is the gap between S phase and M phase. Biology 2101 FG Ayson – Sep 2024  How is DNA Replicated ? Replication occurs in three major steps: 1. The opening of the double helix and separation of the DNA strands. 2. The priming of the template strand. 3. The assembly of the new DNA segment. Biology 2101 FG Ayson – Sep 2024  DNA replication - 2 During separation, the two strands of the DNA double helix uncoil at a specific location called the origin. Several enzymes and proteins then work together to prepare, or prime, the strands for duplication. Finally, the enzyme DNA polymerase organizes the assembly of the new DNA strands. Biology 2101 FG Ayson – Sep 2024  DNA replication - 3 Steps in DNA replication The first step is to “unzip” the double helical structure of DNA. catalyzed by the enzyme helicase which breaks the hydrogen bonds holding the complimentary bases of DNA. The unwinding of the 2 single strands of DNA results in a replication fork. Biology 2101 FG Ayson – Sep 2024  DNA replication - 4 The two separated strands will act as templates for making the new strands of DNA. One of the strands is oriented in the 3’ to 5’ direction (towards the replication fork) and is called the leading strand. The other strand is oriented in the 5’ to 3’ direction (away from the replication fork) and is called the lagging strand. Biology 2101 FG Ayson – Sep 2024  DNA replication - 5 As a result of their different orientations, the 2 strands are replicated differently. In the leading strand A primer binds to the end of the leading strand and acts as the starting point for DNA synthesis. DNA polymerase binds to the leading strand and “walks” along the strand adding new complimentary nucleotides to the strand of the DNA in a 5’ to 3’ direction; replication is continuous. Biology 2101 FG Ayson – Sep 2024  DNA replication - 6 In the lagging strand numerous primers bind at various points along the lagging strand. these act as starting points for the addition of complimentary bases in the 5’ to 3’ direction producing fragments called Okazaki fragments. replication in the lagging strand is discontinuous. Biology 2101 FG Ayson – Sep 2024  DNA replication - 7 In the lagging strand once all the complimentary bases are in place, an enzyme called exonuclease will catalyze the removal of the primer sequences. The gaps are then filled with complimentary bases. The enzyme ligase joins the fragments to make 2 continuous double stranded DNA. The product of DNA replication are 2 DNA molecules consisting of one new and one old chain of nucleotides, hence semi-conservative. Biology 2101 FG Ayson – Sep 2024  DNA replication - 8 Biology 2101 FG Ayson – Sep 2024  DNA replication - 9 Following replication, the new DNA winds up into a double helix. Biology 2101 FG Ayson – Sep 2024  DNA replication - 10 Once the DNA is replicated, the cell has twice the amount of DNA that it needs, and the cell can then divide and parcel this DNA into the daughter cell, so that the daughter cell and the parental cell are absolutely genetically identical. Biology 2101 FG Ayson – Sep 2024  What Triggers Replication ? The initiation of DNA replication occurs in two steps. An initiator protein unwinds a short stretch of the DNA double helix. Then, a protein known as helicase attaches to and breaks apart the hydrogen bonds between the bases on the DNA strands, thereby pulling apart the 2 strands. As the helicase moves along the DNA molecule, it continues breaking these Helicase (yellow) unwinds the hydrogen bonds and separating double helix. the 2 polynucleotides chains. Biology 2101 FG Ayson – Sep 2024  what triggers replication ? - 2 As the helicase separates the strands, another enzyme called primase briefly attaches to each strand and assembles a foundation at which replication can begin. This foundation is a short stretch of nucleotides called a primer. While helicase and the initiator protein separate the two polynucleotide chains, primase (red) assembles a primer. This primer permits the next step in the replication process. Biology 2101 FG Ayson – Sep 2024  what triggers replication ? - 3 primer elongation Biology 2101 FG Ayson – Sep 2024  How is DNA strand replicated ? After the primer is in place in the separated strand, DNA polymerase wraps itself around that strand, and it attaches new nucleotides to the exposed nitrogenous bases. In this way, the polymerase assembles a new DNA strand. Beginning at the primer sequence, DNA polymerase (blue) attaches to the original DNA strand and begins assembling a new, complementary strand. Biology 2101 FG Ayson – Sep 2024  how is DNA strand replicated ? - 2 DNA polymerase relies upon the pool of free-floating nucleotides surrounding the existing strand to build the new strand by complementary base pairing and it results in the production of two complementary strands of DNA. Biology 2101 FG Ayson – Sep 2024  Types of DNA Polymerases In prokaryotes, 3 types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair. Eukaryotic cells contain five DNA polymerases: α, β, γ, δ, and ε. DNA polymerase γ is located in mitochondria and is responsible for replication of mitochondrial DNA. The other 4 are located in the nucleus. DNA polymerases α and δ function for DNA replication. Biology 2101 FG Ayson – Sep 2024  DNA polymerases - 2 DNA polymerases share two fundamental properties that carry critical implications for DNA replication. (1) all polymerases synthesize DNA only in the 5′ to 3′ direction, adding a dNTP to the 3′ hydroxyl group of a growing chain. (2) DNA polymerases can add a new deoxyribonucleotide only to a preformed primer strand that is hydrogen-bonded to the template; they are not able to initiate DNA synthesis de novo by catalyzing the polymerization of free dNTPs. Biology 2101 FG Ayson – Sep 2024  DNA polymerases - 3 In this respect, DNA polymerases differ from RNA polymerases , which can initiate the synthesis of a new strand of RNA in the absence of a primer. These properties of DNA polymerases appear critical for maintaining the high fidelity of DNA replication that is required for cell reproduction. Biology 2101 FG Ayson – Sep 2024  The Reaction Catalyzed by DNA Polymerase DNA polymerases add a deoxyribonucleoside 5′-triphosphate (dNTP) to the 3′ hydroxyl group of a growing DNA chain (the primer strand). Biology 2101 FG Ayson – Sep 2024  Replication Fork The synthesis of new DNA strands complementary to both strands of the parental molecule posed an important problem to understanding the biochemistry of DNA replication. Since the two strands of double-helical DNA run in opposite (anti-parallel) directions, continuous synthesis of two new strands at the replication fork would require that one strand be synthesized in the 5′ to 3′ direction while the other is synthesized in the opposite (3′ to 5′) direction. But DNA polymerase catalyzes the polymerization of dNTPs only in the 5′ to 3′ direction. How then, can the other strand of DNA be synthesized? Biology 2101 FG Ayson – Sep 2024  replication fork - 2 This was resolved by experiments showing that only one strand of DNA is synthesized in a continuous manner in the direction of overall DNA replication. The other strand is formed from small, discontinuous pieces of DNA that are synthesized backward with respect to the direction of movement of the replication fork. These small pieces of newly synthesized DNA (called Okazaki fragments) are joined by the action of DNA ligase, forming an intact new DNA strand. Biology 2101 FG Ayson – Sep 2024  Synthesis of Leading and Lagging Strands of DNA The leading strand is synthesized continuously in the direction of replication fork movement. The lagging strand is synthesized in small pieces (Okazaki fragments) backward from the overall direction of replication. Biology 2101 FG Ayson – Sep 2024  synthesis of leading and lagging strands of DNA - 2 Biology 2101 FG Ayson – Sep 2024  Origin and Initiation of Replication The replication of both prokaryotic and eukaryotic DNAs starts at a unique sequence called the origin of replication which serves as a specific binding site for proteins that initiate the replication process. In E. coli, replication always begins at a unique site and found to consist of 245 base pairs of DNA. The key step is the binding of an initiator protein to specific DNA sequences within the origin. The initiator protein begins to unwind the DNA. Biology 2101 FG Ayson – Sep 2024  origin and initiation of replication - 2 Helicase and single-stranded DNA-binding proteins then act to continue unwinding and exposing the template DNA. Primase initiates the synthesis of leading strands. Two replication forks are formed and move in opposite directions along the circular E. coli chromosome. Biology 2101 FG Ayson – Sep 2024  origin and initiation of replication - 3 DNA rich in A-T base pairs is relatively easy to pull apart, and regions of DNA enriched in A-T pairs are typically found at replication origins. Biology 2101 FG Ayson – Sep 2024  Model for Bacterial DNA Replication Initiation Circular bacterial chromosomes contain a replicator, that is located at or near replication origins. i) The replicator recruits initiator proteins in a DNA sequence-specific manner, which results in melting of the DNA helix and loading of the helicase onto each of the single DNA strands (ii). iii) Assembled replisomes bidirectionally replicate DNA to yield two copies of the bacterial chromosome. Biology 2101 FG Ayson – Sep 2024  Prokaryotic DNA Replication Replication of DNA in prokaryotes begins at a single origin of replication and proceeds in a bidirectional manner around the circular chromosome until replication is complete. The bidirectional nature of replication creates two replication forks that are actively mediating the replication process. Biology 2101 FG Ayson – Sep 2024  prokaryotic DNA replication - 2 The dynamic model of the process. The red and blue dots represent the incorporation of daughter strand nucleotides during the process of replication. Biology 2101 FG Ayson – Sep 2024  prokaryotic DNA replication - 3 DNA replication of a bacterial genome. It takes E. coli about 40 minutes to duplicate its genome of 4.6 × 106 nucleotide pairs. (For simplicity, no Okazaki fragments are shown on the lagging strand). Biology 2101 FG Ayson – Sep 2024  prokaryotic DNA replication - 4 The proteins that initiate DNA replication in bacteria. Biology 2101 FG Ayson – Sep 2024  Eukaryotic DNA Replication In eukaryotic cells, DNA synthesis is initiated at multiple sites, from which it then proceeds in both directions. The replication origins in mammalian cells are spaced at intervals of approximately 50 to 300 kb; thus, the human genome has about 30,000 origins of replication. The genomes of simpler eukaryotes also have multiple origins; for example, replication in yeasts initiates at origins separated by intervals of approximately 40 kb. Biology 2101 FG Ayson – Sep 2024  How Long Does Replication Take ? In the prokaryotic bacterium E. coli, replication can occur at a rate of 1,000 nucleotides per second. In comparison, eukaryotic human DNA replicates at a rate of 50 nucleotides per second. In both cases, replication occurs so quickly because multiple polymerases can synthesize two new strands at the same time by using each unwound strand from the original DNA double helix as a template. Biology 2101 FG Ayson – Sep 2024  origin and initiation of replication - 4 Single origins are sufficient to direct the replication of bacterial and viral genomes. For example, the entire genome of E. coli (4 × 106 base pairs) is replicated from a single origin in approximately 30 minutes. Multiple origins are needed to replicate the much larger genomes of eukaryotic cells within a reasonable period of time. If mammalian genomes (3 × 109 base pairs) are replicated from a single origin at the same rate, DNA replication would require about 3 weeks (30,000 minutes). Biology 2101 FG Ayson – Sep 2024  origin and initiation of replication - 5 The problem is further exacerbated by the fact that the rate of DNA replication in mammalian cells is actually about tenfold lower than in E. coli, possibly as a result of the packaging of eukaryotic DNA in chromatin. As such, the genomes of mammalian cells are typically replicated within a few hours, necessitating the use of thousands of replication origins. Biology 2101 FG Ayson – Sep 2024  Replication Origins in Eukaryotes Replication initiates at multiple origins (ori), each of which produces two replication forks. Biology 2101 FG Ayson – Sep 2024  Model for Eukaryotic DNA Replication Initiation Linear eukaryotic chromosomes contain many replication origins. Initiator binding (i) facilitates helicase loading (ii) onto duplex DNA to license origins. iii) A subset of loaded helicases is activated for replisome assembly. Replication proceeds bidirectionally from origins and terminates when replication forks from adjacent active origins meet (iv). Biology 2101 FG Ayson – Sep 2024  Fidelity of Replication The accuracy of DNA replication is critical to cell reproduction. Estimates of the frequency of errors during replication corresponds to only one incorrect base per 109 to 1010 nucleotides incorporated. The higher degree of fidelity results largely from the activities of DNA polymerase. Biology 2101 FG Ayson – Sep 2024  How Fidelity in Replication is Achieved ? DNA polymerase selects the correct base for insertion into newly synthesized DNA. The polymerase does not simply catalyze incorporation of whatever nucleotide is hydrogen-bonded to the template strand, it actively discriminates against incorporation of a mismatched base, presumably by adapting to the conformation of a correct base pair. The molecular mechanisms responsible for the ability of DNA polymerases to select against incorrect bases are not yet entirely understood, but this selectivity appears to increase the accuracy of replication about a hundred-fold, reducing the expected error frequency from 10-4 to approximately 10-6. Biology 2101 FG Ayson – Sep 2024  fidelity in replication - 2 DNA polymerase has a proof-reading activity. E. coli DNA polymerase I has 3′ to 5′ as well as 5′ to 3’ exonuclease activity. The 5′ to 3′ exonuclease operates in the direction of DNA synthesis and helps remove RNA primers from Okazaki fragments. The 3′ to 5′ exonuclease operates in the reverse direction of DNA synthesis and participates in proof-reading newly synthesized DNA. Biology 2101 FG Ayson – Sep 2024  fidelity in replication - 3 Proof-reading is effective because DNA polymerase requires a primer and is not able to initiate synthesis de novo. Primers that are hydrogen-bonded to the template are preferentially used, so when an incorrect base is incorporated, it is likely to be removed by the 3′ to 5′ exonuclease activity rather than being used to continue synthesis. Biology 2101 FG Ayson – Sep 2024  fidelity in replication - 4 The 3′ to 5′ exonuclease activities are associated with E. coli polymerase III and eukaryotic polymerases δ and ε. The 3′ to 5′ exonucleases of these polymerases selectively excise mismatched bases that have been incorporated at the end of a growing DNA chain, thereby increasing the accuracy of replication by a hundred- to a thousand-fold. Biology 2101 FG Ayson – Sep 2024  General Overview of a DNA Replication Fork At the origin of replication, topoisomerase II relaxes the supercoiled chromosome. Two replication forks are formed by the opening of the double-stranded DNA at the origin, and helicase separates the DNA strands. DNA replication occurs in both directions. An RNA primer complementary to the parental strand is synthesized by RNA primase and is elongated by DNA polymerase III through the addition of nucleotides to the 3′-OH end. Biology 2101 FG Ayson – Sep 2024  General Overview of a DNA Replication Fork On the leading strand, DNA is synthesized continuously. On the lagging strand, DNA is synthesized in short stretches called Okazaki fragments. RNA primers within the lagging strand are removed by the exonuclease activity of DNA polymerase I, and the Okazaki fragments are joined by DNA ligase. Biology 2101 FG Ayson – Sep 2024  General Overview of a DNA Replication Fork Biology 2101 FG Ayson – Sep 2024  Overview of the DNA Replication Process https://youtu.be/TNKWgcFPHqw?si=PkGvCrQ7R q9UPl_B Biology 2101 FG Ayson – Sep 2024