Molecular Genetics Lecture 03 - DNA Replication and Mutation PDF

Summary

This lecture covers the process of DNA replication, including the differences between prokaryotic and eukaryotic replication, as well as various types of mutations.

Full Transcript

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

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