DNA Replication in Bacteria - PowerPoint

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					   UNIT 2

DNA Replication
 Discuss experimental evidence supporting semi-
   conservative mechanism of DNA replication
 Explain DNA replication in Prokaryotes and
 Define mutation

 Identify different types of mutations and their
   effects on the protein products produced
   Eukaryotic genes have interrupted coding sequences.

   That is, there are long sequences of bases within the
    protein-coding sequences of the gene that do not code
    for amino acids in the final protein product.

   The nocoding regions within the gene are called introns
    (intervening sequences).

   The exons (expressed sequences) which are part of the
    protein-coding sequence.
   A typical eukaryotic gene may have multiple exons and
    introns and the numbers are quite variable.

    Eg. the β-globulin gene has 2 and the ovalbumin gene
    of egg white has 7.

   In many cases the lengths of the introns are much
    greater than those of the exon sequences.

   For instance the ovalbumin gene contains about 7700
    base pairs, 1859 of them in exons.
                 DNA Replication
Three theories were suggested:

   Conservative replication
       intact the original DNA molecule and generate a completely
        new molecule.

   Dispersive replication
       produce two DNA molecules with sections of both old and
        new DNA interspersed along each strand.

   Semi-conservative replication
       produce molecules with both old and new DNA - each
        molecule would be composed of one old strand and one new
    DNA Replication is semi-conservative
          Experimental Proof

   (1957) Mathew Meselson and Franklin Stahl grew the
    bacterium Escherichia coli on medium that contained 15N
    in the form of ammonium chloride.

   The 15N became incorporated into DNA (nitrogenous

   The resulting heavy nitrogen-containing DNA
    molecules were extracted from some of the cells.
    DNA Replication is semi-conservative
          Experimental Proof
   When subject to density gradient centrifugation, they
    accumulated in the high-density region of the gradient.

   The rest of the bacteria were transferred to a new
    growth medium in which ammonium chloride
    contained the naturally abundant, lighter 14N isotope.
    DNA Replication is semi-conservative
          Experimental Proof
   The newly synthesized strands were expected to be less
    dense since they incorporated bases containing the
    lighter 14N isotope.

   The DNA from cells isolated after one generation had
    an intermediate density, indicating that they contained
    half as many 15N isotope as the parent DNA.

   This finding supported the semi-conservative model -
    each double helix would contain one previously
    synthesized strand and a newly synthesized strand.
    DNA Replication is semi-conservative
          Experimental Proof
   It is also consistent with the dispersive model which
    would yield one class of molecules, all with intermediate

   It was inconsistent with the conservative model which
    predicted that there would be two classes of double-
    stranded molecules, those with two heavy strands and
    those with two light strands.

   After another cycle of cell division in the medium with
    the lighter 14N isotope, two types of DNA appeared in
    the density gradient.
    DNA Replication is semi-conservative
          Experimental Proof
   One with hybrid DNA helices ( one strand 15N isotope
    and the other strand 14N), whereas the other contained
    only strands of the light isotope.

   This finding refuted the dispersive model, which predicted
    that all stands should have intermediate density.

   It however supported the semiconservative method which
    predicted that each parent strand would act as a template
    for the synthesis of new strands.
      Animation of DNA Replication
              Experimental Proof

   http://highered.mcgraw-

     DNA Replication in Bacteria
   In general, DNA is replicated by:
     uncoiling of the helix
     strand separation by breaking of the hydrogen
      bonds between the complementary strands
     synthesis of two new strands by complementary
      base pairing

    Replication begins at a specific site in the DNA called
     the origin of replication (ori)
    DNA Replication in Bacteria
   DNA replication is bidirectional from the
    origin of replication
   DNA replication occurs in both directions from
    the origin of replication in the circular DNA
    found in most bacteria.
    DNA Replication in Bacteria
   To begin DNA replication, unwinding enzymes
    called DNA helicases cause the two parent
    DNA strands to unwind and separate from one
    another at the origin of replication to form two
    "Y"-shaped replication forks.

   These replication forks are the actual site of
    DNA copying
Replication Fork
    Animation of Replication Fork
   http://highered.mcgraw-
    DNA Replication in Bacteria
   Helix destabilizing proteins bind to the
    single-stranded regions so the two strands do
    not rejoin
   Enzymes called topoisimerases produce breaks
    in the DNA and then rejoin them in order to
    relieve the stress in the helical molecule during
    DNA Replication in Bacteria
   As the strands continue to unwind in both directions
    around the entire DNA molecule, new
    complementary strands are produced by the
    hydrogen bonding of free DNA nucleotides with
    those on each parent strand

   As the new nucleotides line up opposite each parent
    strand by hydrogen bonding, enzymes called DNA
    polymerases join the nucleotides by way of
    phosphodiester bonds.
    DNA Replication in Bacteria
   The nucleotides lining up by complementary
    base pairing are deoxynucleoside triphosphates

    As the phosphodiester bond forms between the
    5' phosphate group of the new nucleotide and
    the 3' OH of the last nucleotide in the DNA
    strand, two of the phosphates are removed
    providing energy for bonding
DNA Replication by Complementary
          Base Pairing
    Animation of How Nucleotides are
   http://highered.mcgraw-
DNA replication in a 5' to 3'
    DNA Replication in Bacteria
   DNA replication is more complicated than this because
    of the nature of the DNA polymerases.

    DNA polymerase enzymes are only able to join the
    phosphate group at the 5' carbon of a new
    nucleotide to the hydroxyl (OH) group of the 3'
    carbon of a nucleotide already in the chain.

   As a result, DNA can only be synthesized in a 5' to 3'
    direction while copying a parent strand running in a 3'
    to 5' direction.
    DNA Replication in Bacteria
   The two strands are antiparallel –
       one parent strand - the one running 3' to 5' is called
        the leading strand can be copied directly down its
        entire length

       the other parent strand - the one running 5' to 3' is
        called the lagging strand must be copied
        discontinuously in short fragments –

        Okazaki fragments of around 100-1000 nucleotides
        each as the DNA unwinds.
         DNA Replication in Bacteria
   DNA polymerase enzymes cannot begin a new
    DNA chain from scratch.

       can only attach new nucleotides onto 3' OH group of a
        nucleotide in a preexisting strand.

       To start the synthesis of the leading strand and each DNA
        fragment of the lagging strand, an RNA polymerase
        complex called a primosome or primase is required.

       The primase is capable of joining RNA nucleotides without
        requiring a preexisting strand of nucleic acid - forms what is
        called an RNA primer
RNA primer
DNA Replication in Bacteria
   After a few nucleotides are added, primase is replaced
    by DNA polymerase.

   DNA polymerase can now add nucleotides to the 3'
    end of the short RNA primer.

   The primer is later degraded and filled in with DNA.
        DNA Replication in Bacteria
Bacteria have 5 known DNA polymerases:
   Pol I:

     DNA repair
     has 5'→3' (Polymerase) activity

     both 3' → 5' (proof reading) and 5' → 3' exonuclease
      activity (in removing RNA primers).
DNA polymerase I is not the replicative polymerase:

   1. The enzyme is too slow!
     adds dNTPs at a rate of 20 nt/sec. So it would require
     460,000 sec (= 7667 min = 128 hr = 5.3 days) to replicate
     the E. coli chromosome! Too slow for an organism which
     can divide every 20 mins.

   2. The enzyme is too abundant
     There are 400 molecules per E. coli cell. This is excessive
     given that there are generally only 2 replication forks per

   3.The enzyme is not processive enough
     DNA polymerase I dissociates after catalysing the
     incorporation of 20-50 nucleotides.
        DNA Replication in Bacteria
   Pol II:
     involved in repair of damaged DNA
     has 3' → 5' exonuclease activity.

Proof that this is not the main polymerase:
1. Strains lacking the gene show no defect in growth or

2. Synthesis of Pol II is induced during the stationary phase
   of cell growth - a phase in which little growth and DNA
   synthesis occurs. But DNA can accumulate damage such
   as short gaps

3. Pol II has a low error rate but it is much too slow to be
   of any use in normal DNA synthesis.
    DNA Replication in Bacteria
   Pol III:
       the main polymerase in bacteria (elongates in
        DNA replication)
       has 3' → 5' exonuclease proofreading ability.
   is the principal replicative enzyme

Proof of function:
1. is highly processive
2. catalyses polymerization at a high rate.
   There are two forms of the enzyme.
    Core enzyme - consists of only those subunits that
    are required for the basic underlying enzymatic
    activity: alpha (a), epsilon (e) and theta (q).

    Holoenzyme- the fully functional form of an
    enzyme, complete with all of its necessary
    accessory subunits.
    The DNA polymerase III holoenzyme consists of
    the core enzyme, the b sliding clamp and the
    clamp-loading complex.
    DNA Replication in Bacteria
   Pol IV and Pol V:

     Are Y-family DNA polymerases
     participates in bypassing DNA damage
DNA Replication in Bacteria
        Animation of bidirectional
           replication of DNA
   http://highered.mcgraw-
     DNA Replication in Eukaryotes
   multiple origins of replication in eukaryotes
       human genome about 30,000 origins
   each origin produces two replication forks
       moving in opposite direction
DNA Replication in Eukaryotes
    DNA Replication in Eukaryotes
Eukaryotes have at least 15 DNA Polymerases:

   Pol α : act as a primase (synthesizing an RNA primer),
    elongates the primer
   Pol β : repairs DNA, (excision repair and gap-filling).
   Pol γ: Replicates and repairs mitochondrial DNA and has
    proofreading 3' → 5' exonuclease activity.
   Pol δ: Highly processive and has proofreading 3' → 5'
    exonuclease activity, reposible for replication of lagging
   Pol ε: Highly processive and has proofreading 3' → 5'
    exonuclease activity, reponsible for replication of leading
   η, ι, κ, Rev1 and Pol ζ are involved in the bypass of DNA
   θ, λ, φ, σ, and μ are not as well characterized:
   There are also others, but the nomenclature has become
    quite jumbled.
        DNA Replication in Eukaryotes
   the polymerases that deal with the elongation are
     Pol α, Pol ε,Polδ.

   Pol α : forms a complex to act as a primase
    (synthesizing an RNA primer), and then elongates that
    primer with DNA nucleotides.

   After around 20 nucleotides elongation by Pol α is taken
    over by Pol ε (on the leading strand) and δ (on the
    lagging strand).

   Other enzymes are responsible for primer remover in
    Eukaryotes as none of their polymerases have 5′→3′
    exonuclease activity
DNA Replication in Eukaryotes
               DNA damage bypass
   All organisms need to deal with the problems that arise
    when a moving replication fork encounters damage in the
    template strand.

   The best way to deal with this situation is to repair the
    damage by an excision mechanisms.

   In some cases, however, the damage may not be repairable,
    or the advancing replication fork may already have
    unwound the parental strands, thus preventing excision
    mechanisms from using the complementary strand as
    template for repair, or excision repair may not yet have had
    an opportunity to repair the damage.
           DNA damage bypass
   It is important for the cell to be able to move
    replication forks past unrepaired damage:
      Long-term blockage of replication forks leads
        to cell death.
      Replication of damaged DNA provides a sister
        chromatid that can be used as template for
        subsequent repair by homologous
   Replication fork bypass mechanisms cannot,
    strictly speaking, be considered examples of DNA
    repair, because the damage is left in the DNA, at
    least temporarily.
           Rate of Replication
   In prokaryotes replication proceeds at about 1000
    nucleotides per second, and thus is done in no more
    than 40 minutes.

   In Eukaryotes replication takes proceeds at 50
    nucleotides per second, and is completed in 60
   changes in the nucleotide sequence of the DNA.

   organisms have special systems of enzymes that can
    repair certain kinds of alterations in the DNA.

   once the DNA sequence has been changed, DNA
    replication copies the altered sequence just as it would
    copy a normal sequence.

   provide the variation necessary for evolution to happen
    in a given species.
              Types of Mutations
Somatic mutations
   Occurs in cells not dedicated to sexual reproduction

   The mutant genes disappear when the cell in which it
    occurred dies and can only be passed on through
    asexual reproduction.

Germline mutations
   found in every cell descended from the zygote to which
    that mutant gamete contributed.

   If an adult is successfully produced, every one of its cells
    will contain the mutation.
            Types of Mutations
Single-base Substitution/point mutation
 exchanges one base for another.

    If one purine [A or G] or pyrimidine [C or T] is
     replaced by the other, the substitution is called a
      If a purine is replaced by a pyrimidine or vice-versa,
       the substitution is called a transversion.

  Original:      The fat cat ate the wee rat
  Point Mutation: The fat hat ate the wee rat
          Types of Mutations
Point mutations continued
 A change in a codon to one that encodes a
   different amino acid and cause a small change in
   the protein produced = missense mutation.
     Example sickle-cell disease
     A → T at the 17th nt of the gene for the beta
     chain of hemoglobin changes the codon GAG
     (glutamic acid) to GTG (valine)
Therefore: 6th amino acid glutamic acid → valine
Missense mutation
     Examples of Diseases caused by
           point mutations
   Color blindness
   Cystic fibrosis
   Hemophilia
   Phenylketonuria
   Tay Sachs
                Types of Mutations
Point mutations continued
 change a codon to one that encodes the same amino
  acid and causes no change in the protein produced
  = silent mutations.

   change an amino-acid-coding codon to a single
    "stop" codon → an incomplete protein
    = a nonsense mutation
       can have serious effects since the incomplete protein
        probably won't function.
Nonsense Mutation
               Types of Mutations

   extra base pairs are inserted into a new place in the DNA.
    Original: The fat cat ate the wee rat.
    Insertion: The fat cat xlw ate the wee rat.


   a section of DNA is lost, or deleted.
    Original: The fat cat ate the wee rat.
    Deletion: The fat      ate the wee rat.
Insertion mutation
            Types of Mutations

   An example of a human disorder caused by insertion is
    Huntington’s disease.

   In this disorder, the repeated trinucleotide is CAG,
    which adds a string of glutamines (Gln) to the encoded
    protein (called huntingtin).

   The abnormal protein increases the level of the p53
    protein in brain cells causing their death by apoptosis.
Deletion Mutation
     Examples of Diseases caused by
   Cri du chat
   De Grouchy syndrome
   Shprintzen syndrome
   Wolf-Hirschhorn syndrome
   Duchenne muscular dystrophy
                   Types of Mutations
   Insertion and deletions involving one or two base pairs (or
    multiples )
      can have devastating consequences to the gene because
       translation of the gene is "frameshifted"
       DNA is read in sequences of three bases therefore the addition or removal of
        one or more bases alters the sequence that follows as the bases all shifted.

       The entire meaning of the sequence has changed.

   Frameshifts often create new STOP codons → nonsense
    Original: The fat cat ate the wee rat.
    Frame Shift: The fat caa tet hew eer at.
Frame shift mutation
            Types of Mutations

   Duplications are a doubling of a section of the

   During meiosis, crossing over between sister
    chromatids that are out of alignment can produce one
    chromatid with an duplicated gene and the other
    having two genes with deletions.
       Example of disease :DM1 (Myotonic dystrophy)
              Types of Mutations

   Translocations are the transfer of a piece of one
    chromosome to a nonhomologous chromosome.

   Translocations are often reciprocal; that is, the two
    nonhomologues swap segments.
               Types of Mutations
Translocations can alter the phenotype is several ways:

   the break may occur within a gene
       destroying its function
       creating a hybrid gene.

   translocated genes may come under the influence of
    different promoters and enhancers so that their
    expression is altered.
             Types of Mutations

   an entire section of DNA is reversed.

   A small inversion may involve only a few bases within
    a gene, while longer inversions involve large regions of
    a chromosome containing several genes.

    Original: The fat cat ate the wee rat.
    Insertion: The fat tar eew eht eta tac.
              Types of Mutations
Suppressor mutation

   partially or completely masks phenotypic expression of
    a mutation but occurs at a different site from it
    (i.e., causes suppression)
    may be intragenic or intergenic.

   It is used particularly to describe a secondary mutation
    that suppresses a nonsense codon created by a primary
                     Naming genes
   given an official name and symbol by a formal committee
   The HUGO Gene Nomenclature Committee (HGNC) – US
    and UK designates an official name and symbol (an abbreviation
    of the name) for each known human gene.
   Some official gene names include additional information in
    parentheses, such as related genetic conditions, subtypes of a
    condition, or inheritance pattern.
   The Committee has named more than 13,000 of the estimated
    20,000 to 25,000 genes in the human genome.
   a unique name and symbol are assigned to each human gene,
    which allows effective organization of genes in large databanks,
    aiding the advancement of research.
    How are genetic conditions named?
Disorder names are often derived from one or a combination of
 The basic genetic or biochemical defect that causes the condition
  (alpha-1 antitrypsin deficiency)
   One or more major signs or symptoms of the disorder (sickle cell
   The parts of the body affected by the condition (retinoblastoma)
   The name of a physician or researcher, often the first person to
    describe the disorder (Marfan syndrome - Dr. Antoine Marfan)
   A geographic area (familial Mediterranean fever)
   The name of a patient or family with the condition (Lou Gehrig
   Disorders named after a specific person or place are called eponyms.
    References/ sources of images

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