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DNA Replication

We all know...


In 1953, James Watson and Francis Crick published their two-page paper (in Nature) entitled: "Molecular structure of Nucleic Acids: A Structure for Deoxyribonucleic Acid".

Their model of DNA's structure


Recall:

Note:


The Magic of DNA Replication

In 1958, Matthew Meselson and Francis Stahl published their work on DNA replication. They used a technique they invented, density gradient centrifugation, to demonstrate that DNA replication was semi-conservative.

To do this, they used 15N to label DNA molecules and hybridized the strands in vitro and then allowed their samples to replicate.

They found that DNA replication is semi-conservative, meaning that each newly replicated strand consists of half the original template and half new material.

In 1963, J. Cairns published work on semiconservative replication in E. coli, done via autoradiography.

Recall that prokaryote DNA is circular. During replication, the circle remains unbroken, and DNA replication takes place along the "opened" circle of DNA. The intervening structure on the radiograph resembled the Greek letter "theta",and so Cairns called the structure of the E. coli DNA in mid-replication theta structure.

But are both Y junctions widening (i.e., is replication proceeding in two directions at once?), or is only one moving (i.e., is DNA replication taking place in only one direction?


DNA Synthesis: The Raw Materials

What we will cover initially is what's known from our old pal, E. coli.

What is needed for DNA synthesis? Raw materials for assembly. As you know, the individual monomers of the DNA polymer are nucleotides.

  • nucleoside - any sugar-base compound

  • nucleotide - nucleoside-phosphate ...are the nucleoside triphosphate raw materials of DNA synthesis.

    A deoxyribose nucleotide is manufactured from deoxyribose sugar by binding

    These nucleotides must be joined via phosphodiester bonds (joining 5' to 3' of adjacent nucleotides) to form that famous polymer of nucleotides, a DEOXYRIBONUCLEIC ACID (DNA)

    DNA Replication: The Enzymatic Machinery

    The enzymes that mediate DNA replication in E. coli are...

    As with RNA polymerase, the DNA polymerases are capable of adding new nucleotides only in the 5' to 3' direction. (The 5' end of the new, incoming nucleotide is attached to the 3' end of the nucleotide already attached to the growing strand.)

    Polymerases specifically catalyze 5' to 3' bonding. Result: 2 phosphate groups are lost from the incoming nucleotide, the breaking of those bonds providing energy to drive the replication process.

    Here's an overview of the players in DNA synthesis.


    DNA Replication: Step by Step

    In E. coli:
  • The origin of replication is located at a gene named oriC. The gene is about 245bp long, and is recognized and bound by special initiator proteins.

  • Initiator proteins

  • A primosome is a two-protein complex consisting of

  • The denatured/separated DNA strands, which have an affinity for one another, are prevented from rejoining by helix destabilizing proteins (a.k.a. single-strand binding proteins, or ssbs).

  • Synthesis is bidirectional.

  • Because DNA nucleotides can be laid down only in the 5' to 3' direction, a complication arises at the Y junction:

  • In eukaryotes, an Okazaki fragment will be manufactured at the 3' of any origin of replication on the lagging strand. The 5' end of incoming nucleotides will facing inwards towards the 3' end of the attached nucleotide, as always.

  • Thus, continuous and discontinuous replication are taking place simultaneously on both template strands, in opposite directions:

  • When a circular chromosome such as that of a prokaryote or plasmid replicates, continuous and discontinuous replication also take place on both replicating strands.

  • Plasmids undergo rolling circle replication, in which one entire strand of the double helix is replicated continuously, and the other discontinuously. This allows the plasmid (or virus) to make many copies relatively quickly.


    DNA Replication: A Closer Look at Continuous Replication

    1. a short (2 - 60bp, depending on the organism) primer (consisting of RNA nucleotides) is laid down at the initiation site by the RNA polymerase called primase. The primer has a free 3'-OH end exposed, to which the 5' end of the incoming nucleotide can be attached by DNA polymerase III.

    2. DNA p'ase III continues this process smoothly, adding new nucleotides in the 5' to 3' direction. There are no breaks in the chain until the entire strand is replicated.

    DNA Replication: A Closer Look at Discontinuous Replication


    Topoisomers (topological isomers) of DNA

  • The typical, circular prokaryote chromosome is found in the form of "relaxed" DNA.

  • During replication, DNA can become supercoiled:

    Positive supercoiling can cause problems.

    Enzymes that coil/uncoil topoisomers of DNA are known as topoisomerases. Some of these attach and operate just in front of the Y-junction to prevent tangles in the DNA due to supercoiling as it unwinds.

    There are two types of topoisomerases that act to release the supercoiling and prevent tangling of the DNA strand as it unwinds:

    DNA gyrase is a type II bacterial topoisomerase that catalyzes the ATP-dependent negative super-coiling of the double-stranded, circular chromosome. Because this enzyme is unique to bacteria, it is of interest as a possible target for anti-bacterial agents.
    For example, the fluoroquinolone class of antibiotics (e.g., ciprofloxacin, enrofloxacin, marbofloxacin, etc.) target and inactivate DNA gyrase. The result: when a bacterial pathogen begins to replicate its DNA, the polymer becomes snarled and cannot be copied. The cell dies. Victory! At least until the bacterium devises an enzyme to inactivate the antibiotic.


    DNA Replication: Finishing the Lagging Strand

    Now that we have all these adjacent Okazaki fragments, how do we make them into a single strand of DNA?


    Polymerases can work two ways

    We know that DNA polymerases a add nucleotides in the 5' to 3' direction, but they also can remove them in the 3' to 5' direction. The latter process is known as exonuclease activity.

    RNA primers are removed via exonuclease action.

    Exo- and endonuclease action are also important to the proofreading of the new chain:
    if a DNA polymerase detects an error in the nucleotide added to the new strand, it can snip it out and replace it with the correct one.


    Fun Facts About DNA Polymerases

  • DNA polymerase III is a large, complex enzyme consisting of 17 polypeptide subunits.
  • The polymerization core consists of the subunits

  • The θ subunit causes the two parts of the DNA polymerase to dimerize (i.e., form a two-part holoenzyme).

  • Two β subunits form a "sliding clamp" that hold the enzyme onto the DNA strand and keep it from disengaging. This is critical to the processivity of the molecule.

  • The remaining subunits apparently serve a clamp loader function, helping to attach the β sliding clamps to the DNA polymer.


  • DNA p'ase I: Editing and Filling the Gaps

  • Current models suggest that the polymerization core of this enzyme can sense mismatch errors. This causes the enzyme to "back up" as it moves along the DNA polymer so that the 3' end of the mismatched nucleotide is in the enzyme's exonuclease site.

    snip. replace. continue.

  • The rate of DNA replication differs among organisms. For example...


    DNA Replication in Eukaryotes

    In eukaryotes, there are at least 15 DNA polymerases, each with specific functions. In eukaryotic DNA replication...

  • Special enzymes must be employed to complete replication at the telomeres.

    The Trouble with Telomeres

    Telomeres are long, repeating base sequences at the ends of each chromosome. They are crucial to the life and longevity of the cell.

  • "Capped" and unreactive, they keep the ends of the chromosomes in the cell from accidentally reacting with each other and becoming stuck to each other.

  • The human telomere consists of as many as 2000 repeats of the sequence 5' TTAGGG 3'.

  • Understand the telomere and see the possible link to cancer therapies of the future!

  • DNA Polymerases cannot create telomeres.

  • If nothing is added to the terminal end of newly replicated DNA, then chromosomes become successively shorter with each new replication.

  • Carol Greider and Elizabeth Blackburn showed that telomerase, an enzyme composed of protein and RNA, can maintain chromsome length by adding telomeres to the ends of newly replicated eukaryotic chromosomes.

  • The RNA portion of telomerase contains a base sequence complementary to the terminal DNA repeat (in Tetrahymena, a ciliate, it's TTGGGG)

  • the end of the RNA portion "hangs off" the end of the DNA, and it is here that telomerase adds complementary bases to its own RNA strand.

  • The gap left behind the RNA portion of the telomerase can be filled in by DNA polymerase in the normal fashion.


    Eukaryotic DNA Replication Finale: Histone manufacture and assembly of nucleosomes

  • Histones are manufactured while DNA is being replicated, during S phase of interphase.
  • Histone synthesis ceases shortly before DNA replication is complete.
  • DNA is wound around histones as soon as it is replicated, though there are short regions of histone-free DNA just around the replication fork (where they would get in the way of the process!)
  • No one is yet certain whether nucleosomes are made de novo, or semi-conservatively
  • Nucleosome packaging of DNA may be involved in a process we'll cover later, epigenetic inheritance, the apparent inheritance of acquired characteristics. (Lamarck may not have been completely wrong!)



    Cell Division: A Review

    In mitosis, asexual cell division, one parent cell gives rise to two, genetically identical daughter cells.

    mito - Greek for "thread" (referring to the threadlike appearance of the chromosomes during division)

    sis - Greek for "the act of"

    In meiosis, sexual cell division, one parent cell (2n) divides to produce four haploid (n) daughter cells which are then processed into gametes.

    meio - "less"
    Hence, meiosis is "the act of making less"


    Here's an overview

    Mitosis: Asexual Cell Division

    (First, a video review.)

    Recall general cell characteristics

    Mitosis

    Interphase Gap 1, synthesis, Gap 2.

    As we'll see in more detail later, G1 is important in cancer studies. During late G1, cells pass through a "G1 checkpoint". Depending on the conditions, the cell with either go into suspended animation (G0) or into normal mitosis.

    Cancer cells often have a defect in this checkpoint, and rather than going into G0 or mitosis, they enter a state of immortal proliferation, leading to cancer.


  • Initiation of cell division is controlled by the relative concentrations of two proteins in the cell
  • cell division cycle protein #2 (cdc2)
    (This one tends to have a constant concentration)
  • cyclin protein
    (degraded during part of interphase, and so has variable concentration).

  • When cyclin concentration is high,it combines with cdc2 to form a complex known as CdkG1-cyclin complex.

  • When this complex is present in a critical concentration, cell division is initiated.


  • Another side note on cancer:


  • Some species reproduce via parthenogenesis ("virgin birth").

  • a clone is a group of genetically identical organisms.

    In times of stress, even species that ordinarily reproduce asexually may revert to sexual reproduction. (Why might this tendency be adaptive, in evolutionary terms?)


    Meiosis: Sexual Cell Division

    Why Sex?

    The word comes from the Latin secare, which means to cut or divide something that was once whole.

    In heterogamous species, male gametes are defined as those that are small and motile, and female gametes are defined as those that are larger and non-motile

    In isogamous species, the gametes are physically indistinguishable in both mating types, usually designated as "+" and "-".


    Meiosis I - Reduction Division
    Meiosis II - Equational (mitotic) Division

    First, Meiosis I:

  • Prophase I

  • Metaphase I - spindle fibers arrange homologs along the metaphase plate at the cell's equator.

  • Anaphase I - spindle fibers separate homologs, carrying them to opposite poles, but sister chromatids are still connected at the centromere.

  • Telophase I (if it occurs; some species skip this step) is a backwards progression to the interphase-like conditions. In Meiosis, it is termed interkinesis.


    Meiosis II: the equational division is physically the same as mitosis, though the genetics of the cells are different.


    After meiosis, gametogenesis occurs to make the new haploid cells into gametes.

    The generalized animal scenario:

    MALE:

    FEMALE:


    Plant gametogenesis is a bit different:

    MALE:

    FEMALE:


  • polyspermy: fusion of more than one sperm with a single egg.

  • euploidy - the normal number of chromosome sets expected in a given species. (from the Greek eu, meaning "true")

  • aneuploidy - more or fewer than the normal number of chromosomes per homologous pair (from the Greek an - "away" )