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    DNA Replication and Synthesis: The Flow of Biological Information

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    Renz L. Salumbre
    This document summarizes key aspects of DNA replication and synthesis. It discusses (1) semiconservative replication in prokaryotes and eukaryotes, (2) DNA polymerases and their roles, and (3) differences in replication between prokaryotes and eukaryotes. In prokaryotes, replication initiates at oriC and proceeds bidirectionally. In eukaryotes, multiple origins of replication allow faster replication and replication of larger genomes. DNA polymerases synthesize DNA through elongation while maintaining high fidelity through proofreading. Telomerase prevents shortening of chromosomes during eukaryotic replication.

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    DNA Replication and Synthesis: The Flow of Biological Information

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    Renz L. Salumbre
    413 views12 pages
    This document summarizes key aspects of DNA replication and synthesis. It discusses (1) semiconservative replication in prokaryotes and eukaryotes, (2) DNA polymerases and their roles, and (3) differences in replication between prokaryotes and eukaryotes. In prokaryotes, replication initiates at oriC and proceeds bidirectionally. In eukaryotes, multiple origins of replication allow faster replication and replication of larger genomes. DNA polymerases synthesize DNA through elongation while maintaining high fidelity through proofreading. Telomerase prevents shortening of chromosomes during eukaryotic replication.

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    Genetics Lecture Handouts

    Original Title

    Gene Lecture 10 Replication

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    This document summarizes key aspects of DNA replication and synthesis. It discusses (1) semiconservative replication in prokaryotes and eukaryotes, (2) DNA polymerases and their roles, and (3) differences in replication between prokaryotes and eukaryotes. In prokaryotes, replication initiates at oriC and proceeds bidirectionally. In eukaryotes, multiple origins of replication allow faster replication and replication of larger genomes. DNA polymerases synthesize DNA through elongation while maintaining high fidelity through proofreading. Telomerase prevents shortening of chromosomes during eukaryotic replication.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    413 views12 pages

    DNA Replication and Synthesis: The Flow of Biological Information

    Uploaded by

    Renz L. Salumbre
    This document summarizes key aspects of DNA replication and synthesis. It discusses (1) semiconservative replication in prokaryotes and eukaryotes, (2) DNA polymerases and their roles, and (3) differences in replication between prokaryotes and eukaryotes. In prokaryotes, replication initiates at oriC and proceeds bidirectionally. In eukaryotes, multiple origins of replication allow faster replication and replication of larger genomes. DNA polymerases synthesize DNA through elongation while maintaining high fidelity through proofreading. Telomerase prevents shortening of chromosomes during eukaryotic replication.

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    of 12

    Fundamental Genetics

    Lecture 10

    DNA Replication
    and Synthesis
    John Donnie A. Ramos, Ph.D.
    Dept. of Biological Sciences
    College of Science
    University of Santo Tomas

    The Flow of Biological Information


    Replication

    DNA

    Transcription

    RNA

    Translation

    Protein

    1
    Modes of DNA Replication

    Semiconservative Replication

    2
    Semiconservative Replication in Prokaryotes
    ‰ Mathew Messelson and Franklin Stahl (1958)
    ‰ 15N– heavy isotope of N (contains 1 more
    neutron) compared to 14N
    ‰ 15N has high sedimentation rate in cesium
    chloride compared to 14N

    Semiconservative Replication in Prokaryotes

    ‰ Expected results of the Messelson-Stahl experiment

    3
    Semiconservative Replication in Eukaryotes
    ‰ J. Herbert Taylor, Philip
    Woods, and Walter Hughes
    (1957)
    ‰ Used root tip cells from Vicia
    faba (broad bean)
    ‰ Monitored replication using
    3H-Thymidine to label DNA

    ‰ Used autoradiography to
    determine the incorporation
    of 3H-Thymidine
    ‰ Arrested cells at metaphase
    using colchicine

    Replication of E. coli Plasmid


    ‰ Shown by John Cairns (1981) using
    radioisotopes and radiography
    ‰ Replication starts in a single OriC –
    origin of replication (245 bp)
    ‰ Replication is bidirectional
    ‰ Replication fork – unwound DNA helix
    ‰ Replicon – replicated DNA
    ‰ Ter region – region of replication
    termination

    4
    DNA Synthesis in Microorganisms
    ‰ DNA polymerase I (928 aa) – catalyses the synthesis of DNA in vitro
    (A. Kornberg, 1957)
    ‰ Requirements:
    ‰ Deoxyribonucleoside triphosphates, dNTPs (dATP, dCTP, dGTP, dTTP)
    ‰ DNA template
    ‰ Primer

    Chain Elongation
    ‰ 5’ to 3’ direction of DNA synthesis (requires 3’ end of the DNA template)
    ‰ Each step incorporates free 3’ OH group for further elongation

    ‰ DNA replication using DNA polymerase is of high fidelity (highly


    accurate)
    ‰ With exonuclease activity (proofreading ability)

    5
    DNA Polymerases
    ‰ All 3 types requires a primer
    ‰ Complex proteins (100,000 Da)
    Functions of DNA polymerases
    in vivo
    ‰ DNA Pol I – proofreading;
    removes primers and fills gaps
    ‰ DNA Pol II - mainly involved in
    DNA repair from external
    damage
    ‰ DNA Pol III – main enzyme
    involved in DNA synthesis
    ‰ a holoenzyme (>600,000 Da)
    – forms replisome when
    attached to a replication fork.

    Replication in Prokaryotes
    1. Unwinding of DNA helix
    2. Initiation of DNA synthesis
    3. DNA synthesis proper (elongation)
    4. Sealing gaps
    5. Proofreading and error correction

    6
    Unwinding of DNA Helix
    ‰ Takes place in oriC (245 bp) –
    repeating 9mers and 13mers
    ‰ Function of helicases (Dna A, B, C)
    – requires ATP hydrolysis to break
    hydrogen bonds
    ‰ Initiated by Dna A – binds to 9mers
    ‰ Binding of Dna B and Dna C to
    unwound helix
    ‰ Single-stranded binding proteins
    (SSBPs) – prevents reannealing of
    replication bubble.
    ‰ DNA gyrase (a DNA topoisomerase)
    – relaxes the supercoiling of DNA
    helix

    Initiation of DNA Synthesis

    ‰ Synthesis of RNA primer – 5 to 15 RNA bases complementary to


    the DNA template
    ‰ Catalysed by primase (an RNA polymerase)
    ‰ Pimase does not require free 3’ end to initiate synthesis (not unlike
    DNA polymerase III)
    ‰ Function of primase will be continued by DNA polymerase III.

    7
    DNA Synthesis (Elongation)
    ‰ Function of DNA polymerase III
    ‰ Requires free 3’ end
    ‰ Direction of elongation: 5’ to 3’
    ‰ DNA synthesis is continuous in 3’ to 5’
    DNA strand (leading strand) and
    discontinuous in the 5’ to 3’ DNA strand
    (lagging strand).
    ‰ Okazaki fragments – short DNA
    fragments produced in the lagging
    strand
    ‰ Concurrent synthesis of leading and
    lagging strands occur by using DNA pol
    dimer and by a looping mechanism for
    the lagging strand

    Sealing of Gaps, Proofreading


    and Error Correction
    ‰ DNA polymerase I removes all RNA bases produced
    by primase (creates gaps in the lagging strand) and
    replaces it with DNA bases (U to T).
    ‰ DNA ligase seals the gaps by forming
    phosphodiester bonds
    ‰ Exonuclease proofreading (identification of
    mismatched bases) is a function of both DNA
    polemerase I and III (both with 3’-5’ exonuclease
    activity)
    ‰ ε subunit of DNA polymerase III is involved in
    proofreading.
    ‰ Assures high fidelity of DNA replication

    8
    Mutations Affect Replication

    Replication in Eukaryotes
    ‰ Presence of multiple replication origin
    (faster replication, guarantees replication
    of a big genome) – 25K replicons in
    mammalian cells
    ‰ Autonomously replicating sequences
    (ARSs) – origin of replication in yeasts
    (11 bp)
    ‰ Origin site is AT rich region
    ‰ Helicase unwinds double stranded DNA
    and removes histone proteins from DNA
    ‰ Histones reassociates while DNA
    synthesis occurs.

    9
    Eukaryotic DNA Polymerases
    ‰ Pol α - initiates nuclear DNA synthesis
    ‰ 4 subunits (2 acts primase – produces RNA primers)
    ‰ Acts on both leading and lagging strands
    ‰ 2 other subunits continue elongation step (DNA synthesis)
    ‰ Low processivity (short length of synthesized DNA prior to dissociation)
    ‰ Pol δ - replaces Pol α (called polymerase switching)
    ‰ High processivity (during elongation)
    ‰ With 3’-5’ exonuclease activity (proofreading)
    ‰ Pol ε - nuclear DNA synthesis
    ‰ Pol β - DNA repair (the only eukaryotic DNA polymerase with single
    subunit)
    ‰ Pol ξ - DNA repair
    ‰ Pol γ - mitochondrial DNA synthesis (encoded by nuclear gene)

    ‰ Eukaryotes has a high copy number of DNA polymerases (ex. Pol α


    may be up to 50K copies)

    Eukaryotic DNA Replication


    ‰ Telomeres – linear ends of
    eukaryotic chromosomes
    ‰ Problem with lagging
    strand: no 3’ needed by
    DNA polymerase I (after
    removal of RNA primers)
    ‰ Possible result:
    chromosome with shorter
    lagging strand every
    replication step

    10
    Telomerase
    ‰ Enzyme that adds TTGGGG
    repeats on the telomeres (first
    identified in Tetrahymena)
    ‰ Prevents shortening of
    chromosomes
    ‰ Forms a “hairpin loop” on
    chromosome ends using G-G
    bonds
    ‰ Creates a free 3’ on lagging
    strand that can be used by
    DNA polymerase I to replaced
    the removed RNA primer
    ‰ Telomerase is a
    ribonucleoprotein and contains
    RNA sequence (5’ AACCCC 3”-
    serving as template) – reverse
    transcriptase
    ‰ Cleavage of loop after DNA
    synthesis

    DNA
    Recombination
    ‰ Exchange of genetic
    material
    ‰ Homologous
    recombination
    ‰ Ex. Rec A protein
    (produces recB, recC
    and recD genes)

    11
    Gene Conversion
    ‰ Exchange of genetic information between non-homologous
    chromosomes
    ‰ Type of chromosome mutation (recombination)
    ‰ First identified in Neurospora (by Mary Mitchell)
    ‰ Can be repaired but forms recombined genetic material

    12

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