Molecular Genetics Lecture 03 - DNA Replication and Mutation PDF

Genetics (BHS016-1) Dr Taiwo Shittu 1 DNA Replication & Mutations BHS016-1 Topic 3 2 Learning Outcomes ◼ After this topic you should be able to: ❑ Describe the process of DNA replication. ❑ Explain and contrast the replication of the...

Genetics (BHS016-1) Dr Taiwo Shittu 1 DNA Replication & Mutations BHS016-1 Topic 3 2 Learning Outcomes ◼ After this topic you should be able to: ❑ Describe the process of DNA replication. ❑ Explain and contrast the replication of the leading and lagging strands of DNA. ❑ Compare the process of DNA replication in bacteria as compared to eukaryotes. ❑ Name different types of mutation and describe their effects on protein structure and function. 3 DNA Replication Occurs in S-phase 4 Which Model of DNA Replication is Correct? 5 Models of DNA Replication Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 6 The Meselson and Stahl Experiment Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 7 The Meselson and Stahl Experiment Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 8 The Meselson and Stahl Experiment CONCLUSION DNA replication is semi-conservative 9 DNA Replication is: ❑ COMPLEX: helical DNA molecule must untwist while it copies its two anti-parallel strands simultaneously: requires the co-operation of over a dozen enzymes & other proteins. ❑ RAPID: in some prokaryotes, up to 500 nucleotides added per second. It takes only a few hours to copy the 6 billion bases of a single human cell. ❑ ACCURATE: only about one in a billion nucleotides is incorrectly paired. ❑ SEMI-CONSERVATIVE: each new duplex contains one strand of original duplex and one new strand. 10 Replication Origins 11 Replication Origins ◼ DNA replication begins at specific sites called ORIGINS OF REPLICATION. ◼ Double helix denatures (separates into single strands) at origins of replication. ◼ Replication bubble spreads in both directions away from the origin (BI-DIRECTIONAL). ◼ DNA is copied (synthesis) in both directions from origin. 12 Replication Origins Circular DNA replicates by: ◼Theta Replication – (bacterial chromosomes). ◼Rolling Circle Replication – (some plasmids). Linear DNA replicates by: ◼Linear replication – (eukaryotic chromosomes). 13 Bacteria Use Theta Replication Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 14  Bacteria Use Theta Replication One origin of replication. Two replication forks (one moving in each direction). Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 15 Eukaryotes Have Many Origins Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 16 Eukaryotes Use Linear Replication ◼ Replication begins at multiple origins. ◼ DNA synthesis occurs in both directions. ◼ Replication bubbles fuse and join creating two finished DNA molecules. Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 17 Initiation of Replication 18 Initiation In Prokaryotes Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 19 Initiation In Prokaryotes Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 20 Elongation of Replication 21 Elongation In Prokaryotes Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 22 Elongation In Prokaryotes Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 23 Problems of Elongation in Prokaryotes ◼ There are two major problems affecting the elongation stage of DNA replication. ◼ DNA polymerase cannot start a new nucleic acid chain. ◼ DNA polymerase builds DNA 5’->3’, but the two template strands face in opposite directions. 24 Elongation in Prokaryotes ◼ DNA polymerase cannot start a nucleic acid chain (it cannot join the first few nucleotides). ◼ It requires a primer (a short chain of nucleotides) to already be present, and can then add more DNA nucleotides to the end of the primer. ◼ PRIMASE enzyme creates a short RNA chain primer. ◼ DNA polymerase then extends from the 3’ end of the primer to create a new DNA chain. 25 Elongation in Prokaryotes ◼ The problem of starting DNA synthesis… Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 26 Elongation in Prokaryotes ◼ The problem of antiparallel DNA strands… Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 27 Elongation in Prokaryotes Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 28 Elongation in Prokaryotes Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 29 Elongation in Prokaryotes ◼ Primase creates a single primer for LEADING STRANDS. ◼ Primase creates a primer for each OKAZAKI FRAGMENT in the LAGGING STRANDS. Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 30 Elongation in Prokaryotes ◼ DNA polymerase III synthesises DNA. ◼ DNA polymerase I replaces RNA primers later in the process. Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 31 Termination of Replication 32 Termination of Replication Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 33 Termination of Replication Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 34 Termination of Replication Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 35 Summary of Bacterial DNA Replication ◼ Replication begins in replication origins. ◼ Replication proceeds bi-directionally. ◼ Replication forks require: ❑ Helicase, single strand binding proteins, gyrase, primase and DNA polymerase III. ◼ DNA synthesis initiated by short RNA primers. ◼ Synthesis (5’>3’) is continuous on the leading strand but discontinuous on the lagging strand. ◼ Then RNA primers replaced by DNA and all the DNA fragments are joined together. ❑ DNA polymerase I and ligase. 36  Summary of Bacterial DNA Replication You must know the key enzymes and their functions 37 DNA Replication in Eukaryotes 38 Eukaryotic DNA Replication ◼ Very similar to prokaryotic DNA replication. ◼ BUT: ❑ Multiple origins of replication per chromosome. ❑ Require complex co-ordinated activation. ❑ Contain a number of helicases, topoisomerases and single strand binding proteins. ❑ Contain numerous DNA polymerases. 39 Eukaryotic DNA Polymerases ◼ DNA pol epsilon synthesizes leading strand. ◼ DNA pol delta synthesizes lagging strand. ◼ DNA pol alpha synthesizes RNA primers. Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 40 DNA Mutations 41 Fidelity of DNA Replication ◼ DNA Polymerase makes a single nucleotide error every billion (109) nucleotides added. ◼ This high fidelity is due to: ❑ Nucleotide selection. ❑ Proofreading ability. ❑ DNA repair pathways (mismatch repair). 42 Mutations ◼ If an error does occur: ◼ It will most likely occur in non-essential DNA sequence. ◼ But, it may occur within a gene or a regulatory sequence and therefore affect the gene activity or the function of the resulting protein. 43 Mutations ◼ A single base change that occurs relative to a reference is called a single nucleotide polymorphism (SNP) ◼ Depending where the SNPs occur (within genes or not; in which codon position) determines the magnitude of its effect (if any) 44 Mutations – Missense Base Change ◼ If an error occurs within a protein coding region: ◼ It may alter a codon into a different codon that codes for a different amino acid. ◼ This mutation would alter the protein made. Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 45 Mutations – Nonsense Base Change ◼ If an error occurs within a protein coding region: ◼ It may alter a codon into a different codon that codes for no amino acid (Stop codon). ◼ This mutation would truncate the protein made. Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 46 Mutations – Silent Base Change ◼ If an error occurs within a protein coding region: ◼ It may alter a codon into a different codon that codes for the same amino acid. ◼ This mutation would not alter the protein made. Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 47 Mutations – Summary ◼ If an error occurs within a protein coding region: ◼ This mutation would not alter the protein made. Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 48 Mutations – Summary ◼ Why are silent mutations more common in the 3rd position of codons? ◼ WOBBLE-–more likely to code for same a/acid Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 49 Mutations – Summary ◼ Most mutations will not occur in protein-coding gene sequences (recall genome size and how much of nuclear genome is PCGs) ◼ Those that do, a good proportion may not affect the amino acid depending on codon position ◼ Not all a/acid substitutions are equal either ◼ Strongly deleterious (bad) mutations will be selected against quickly in natural populations Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 50 Mutations – Summary ◼ Other mutations can occur (e.g. insertions and deletions—indels) ◼ May affect open-reading frames, resulting in frame shifts ◼ Chromosomal alterations also mutation at a different level Image taken from Pierce, BA. (2012). Genetics: A conceptual approach 51 Summary – You Should Now Know… ◼ The key proteins involved in DNA replication. ◼ The mechanism of continuous DNA synthesis on the leading strand. ◼ The mechanism of discontinuous DNA synthesis on the lagging strand. ◼ The different modes of DNA replication of prokaryotic and eukaryotic chromosomes. ◼ The definition and functional effect of missense, nonsense and silent mutations. 52 To Do List – DNA Replication ◼ Describe theta replication and linear replication. ◼ Explain the process of replication initiation (recognise origin and create replication bubble). ◼ Explain the process of replication elongation (DNA synthesis on leading and lagging strand). ◼ Explain the process of replication termination (remove primers, ligate Okazaki fragments). ◼ Define Okazaki fragments, helicase, primase, polymerase, missense and nonsense mutation. 53 To Do List – DNA Mutation ◼ Explain the difference in the proteins produced if a gene (gene X) contained: ❑ a silent mutation. ❑ or a missense mutation. ❑ or a nonsense mutation. ◼ What might be the effect on protein production if there were a mutation in the promoter of gene X? ◼ What might be the effect on protein production if there were a mutation in the DNA outside of gene X? 54 Before Next Week’s Lecture For lecture next week: ◼ Read about (RNA Structure and Transcription) in a suitable textbook (e.g. Pierce, 4th Ed., Chapter 15). 55