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    Chem 40 April29 Replication

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    Ruth Danielle Gascon
    DNA replication is a semiconservative and bidirectional process that requires unwinding of the DNA helix. The leading strand copies DNA continuously while the lagging strand forms discontinuous fragments called Okazaki fragments that are later joined. DNA polymerase III is the main enzyme for replication in E. coli and adds nucleotides to the 3' end of the growing DNA strand with very high processivity. Eukaryotic DNA replication involves multiple origins of replication and DNA polymerases alpha, delta, and epsilon to completely copy the genome. Telomeres are replicated by the enzyme telomerase to compensate for incomplete replication of chromosome ends. RNA viruses replicate through a DNA intermediate synthesized by the enzyme reverse transcriptase.

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    Chem 40 April29 Replication

    Uploaded by

    Ruth Danielle Gascon
    52 views14 pages
    DNA replication is a semiconservative and bidirectional process that requires unwinding of the DNA helix. The leading strand copies DNA continuously while the lagging strand forms discontinuous fragments called Okazaki fragments that are later joined. DNA polymerase III is the main enzyme for replication in E. coli and adds nucleotides to the 3' end of the growing DNA strand with very high processivity. Eukaryotic DNA replication involves multiple origins of replication and DNA polymerases alpha, delta, and epsilon to completely copy the genome. Telomeres are replicated by the enzyme telomerase to compensate for incomplete replication of chromosome ends. RNA viruses replicate through a DNA intermediate synthesized by the enzyme reverse transcriptase.

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    DNA replication is a semiconservative and bidirectional process that requires unwinding of the DNA helix. The leading strand copies DNA continuously while the lagging strand forms discontinuous fragments called Okazaki fragments that are later joined. DNA polymerase III is the main enzyme for replication in E. coli and adds nucleotides to the 3' end of the growing DNA strand with very high processivity. Eukaryotic DNA replication involves multiple origins of replication and DNA polymerases alpha, delta, and epsilon to completely copy the genome. Telomeres are replicated by the enzyme telomerase to compensate for incomplete replication of chromosome ends. RNA viruses replicate through a DNA intermediate synthesized by the enzyme reverse transcriptase.

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    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    52 views14 pages

    Chem 40 April29 Replication

    Uploaded by

    Ruth Danielle Gascon
    DNA replication is a semiconservative and bidirectional process that requires unwinding of the DNA helix. The leading strand copies DNA continuously while the lagging strand forms discontinuous fragments called Okazaki fragments that are later joined. DNA polymerase III is the main enzyme for replication in E. coli and adds nucleotides to the 3' end of the growing DNA strand with very high processivity. Eukaryotic DNA replication involves multiple origins of replication and DNA polymerases alpha, delta, and epsilon to completely copy the genome. Telomeres are replicated by the enzyme telomerase to compensate for incomplete replication of chromosome ends. RNA viruses replicate through a DNA intermediate synthesized by the enzyme reverse transcriptase.

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    Replication

    (DNA Synthesis)

    Chemistry 40

    How Is DNA Replicated?


    • DNA replication is semiconservative
    • DNA replication is bidirectional
    • Replication requires unwinding of the
    DNA helix
    • DNA replication is semidiscontinuous
    • The lagging strand is formed from
    Okazaki fragments

    Chemistry 40

    1
    Chemistry 40
    Fig. 10-2, p.242

    2
    Features of DNA Replication
    • DNA replication is bidirectional
    – Bidirectional replication involves two
    replication forks, which move in opposite
    directions
    • DNA replication is semi-discontinuous
    – The leading strand copies continuously
    – The lagging strand copies in segments
    (Okazaki fragments) which must be joined

    Chemistry 40

    Bidirectional
    replication of the
    E. coli
    chromosome. (a)
    If replication is
    bidirectional, auto-
    radiograms of
    radioactively
    labeled replicating
    chromosomes
    should show two
    replication forks
    heavily labeled
    with radioactive
    thymidine. (b) An
    autoradiogram of
    the chromosome
    from a dividing E.
    coli cell shows
    bidirectional
    replication. (Photo
    courtesy of David M.
    Prescott, University
    of Colorado.)

    3
    Chemistry 40
    Fig. 10-4, p.244

    Features of Replication
    Mostly in E. coli, but many features are general
    • Replication is bidirectional
    • The double helix must be unwound - by
    helicases
    • Supercoiling must be compensated - by DNA
    gyrase
    • Replication is semidiscontinuous
    • Leading strand is formed continuously
    • Lagging strand is formed from Okazaki
    fragments - discovered by Tuneko and Reiji "O"
    Chemistry 40

    4
    Chemistry 40
    Fig. 10-5a, p.245

    Chemistry 40
    Fig. 10-5b, p.245

    5
    Chemistry 40
    Fig. 10-10, p.249

    Chemistry 40
    Fig. 10-11, p.250

    6
    Chemistry 40

    Chemistry 40
    Table 10-2, p.242

    7
    DNA Polymerase III
    The "real" polymerase in E. coli
    • At least 10 different subunits
    • "Core" enzyme has three subunits - α, ε, and θ
    • Alpha subunit is polymerase
    • Epsilon subunit is 3'-exonuclease
    • Theta subunit is involved in holoenzyme
    assembly
    • The beta subunit dimer forms a ring around
    DNA
    • Enormous processivity - 5 million bases!
    Chemistry 40

    Chemistry 40

    8
    Chemistry 40

    How Is DNA Replicated in Eukaryotic Cells?

    • The cell cycle controls the timing of DNA


    replication
    • Eukaryotic cells contain a number of different
    DNA polymerases
    • DNA polymerase d is the principal DNA
    replicase

    Chemistry 40

    9
    The eukaryotic cell cycle.
    The stages of mitosis and
    cell division define the M
    phase (M for mitosis). G1
    (G for gap, not growth) is
    typically the longest part of
    the cell cycle; G1, is
    characterized by rapid
    growth and metabolic
    activity. Cells that are
    quiescent, that is, not
    growing and dividing (such
    as neurons), are said to be
    in G0. The S phase is the
    time of DNA synthesis. S
    is followed by G2, a
    relatively short period of
    growth when the cell
    prepares for cell division.
    Cell cycle times vary from
    less than 24 hours (rapidly
    dividing cell such as the
    epithelial cells lining the
    mouth and gut) to
    hundreds of days.

    How Are the Ends of Chromosomes


    Replicated?

    • Telomeres, the structures at the ends of


    eukaryotic chromosomes, consist of 5-8 bp
    tandemly repeated G-rich nucleotide
    sequences
    • Telomeres are 1-12 kbp long
    • Telomeres are replicated by an RNA-
    dependent DNA polymerase called
    telomerase

    Chemistry 40

    10
    Telomere replication. (a) In replication of the lagging strand, short RNA primers are added
    (pink) and extended by DNA polymerase. When the RNA primer at the 5′-end of each strand
    is removed, there is no nucleotide sequence to read in the next round of DNA replication.
    The result is a gap (primer gap) at the 5′-end of each strand (only one end of the chromosome
    is shown in this figure). (b) Asterisks indicate sequences at the 3′-end that cannot be copied
    by conventional DNA replication. Synthesis of telomeric DNA by telomerase extends the 5′-
    ends of DNA strands, allowing the strands to be copied by normal DNA replication.

    Eukaryotic DNA Replication


    Like E. coli, but more complex
    • Human cell: 6 billion base pairs of DNA to
    copy
    • Multiple origins of replication: 1 per 3- 300
    kbp
    • DNA polymerase alpha - four subunits,
    polymerase (processivity = 200) but no 3'-
    exonuclease
    • DNA polymerase beta - similar to alpha

    Chemistry 40

    11
    More Eukaryotic polymerases

    • DNA polymerase gamma - DNA-


    replicating enzyme of mitochondria
    • DNA polymerase delta has a 3'-
    exonuclease as well as proliferating cell
    nuclear antigen (PCNA)
    • PCNA give delta unlimited processivity
    and is homologous with prokaryotic pol III
    • DNA polymerase epsilon - highly
    processive, but does not have a subunit
    like PCNA
    Chemistry 40

    How Are RNA Genomes Replicated?

    • Many viruses have genomes composed of


    RNA
    • DNA is an intermediate in the replication of
    RNA viruses
    • The viral RNA serves as a template for DNA
    synthesis
    • The RNA-directed DNA polymerase is called
    Reverse Transcriptase

    Chemistry 40

    12
    Figure 28.16
    The structures of AZT (3′-azido-2′,3′-
    dideoxythymidine). This nucleoside
    was the first approved drug for
    treatment of AIDS. AZT is
    phosphorylated in vivo to give
    AZTTP (AZT 5′-triphosphate), a
    substrate analog that binds to HIV
    reverse transcriptase, HIV reverse
    transcriptase incorporates AZTTP
    into growing DNA chains in place of
    dTTP. Incorporated AZTMP blocks
    further chain elongation because its
    3′-azido group cannot form a
    phosphodiester bond with an
    incoming nucleotide. Host cell DNA
    polymerases have little affinity for
    AZTTP.

    Reverse Transcriptase
    • Primer required, but a strange one - a tRNA
    molecule that the virus captures from the host
    • RT transcribes the RNA template into a
    complementary DNA (cDNA) to form a
    DNA:RNA hybrid
    • All RNA tumor viruses contain a reverse
    transcriptase

    Chemistry 40

    13
    Reverse Transcriptase Activities
    • Three enzyme activities
    – RNA-directed DNA polymerase
    – RNase H activity - degrades RNA in the
    DNA:RNA hybrids
    – DNA-directed DNA polymerase - which makes
    a DNA duplex after RNase H activity destroys
    the viral genome
    • HIV therapy: AZT (or 3'-azido-2',3'-
    dideoxythymidine) specifically inhibits RT

    Chemistry 40

    Chemistry 40
    Table 10-3, p.250

    14

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