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11.9: Major Enzymes

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    The process of DNA replication is catalyzed by a type of enzyme called DNA polymerase (poly meaning many, mer meaning pieces, and –ase meaning enzyme; so an enzyme that attaches many pieces of DNA). Observe Figure 1: the double helix of the original DNA molecule separates (blue) and new strands are made to match the separated strands. The result will be two DNA molecules, each containing an old and a new strand. Therefore, DNA replication is called semiconservative. The term semiconservative refers to the fact that half of the original molecule (one of the two strands in the double helix) is “conserved” in the new molecule. The original strand is referred to as the template strand because it provides the information, or template, for the newly synthesized strand.

    image
    3′ synthesis shown, no enzymes in diagram. ” width=”1024″ height=”508″> Figure 1. By Madprime(Wikipedia) (DNA replication split horizontal) CC BY-SA 2.0
    Diagram of a primer moving along the template strand of DNA.
    Figure 2. Primer and Template

    DNA replication relies on the double-stranded nature of the molecule. One double stranded DNA molecule, when replicated, will become two double-stranded molecules, each containing one original strand and one newly synthesized strand. You remember that the two strands of DNA run antiparallel: one from the 5′ to the 3′, and the other from the 3′ to the 5′. The synthesis of the new DNA strand can only happen in one direction: from the 5′ to the 3′ end. In other words, the new bases are always added to the 3′ end of the newly synthesized DNA strand. So if the new nucleotide is always added to the 3′ end of an existing nucleotide, where does the first nucleotide come from? In fact, DNA polymerase needs an “anchor” to start adding nucleotides: a short sequence of DNA or RNA that is complementary to the template strand will work to provide a free 3′ end. This sequence is called a primer (Figure 2).

    How does DNA polymerase know in what order to add nucleotides? Specific base pairing in DNA is the key to copying the DNA: if you know the sequence of one strand, you can use base pairing rules to build the other strand. Bases form pairs (base pairs) in a very specific way. Figure 3 shows how A (adenine) pairs with T (thymine) and G (guanine) pairs with C (cytosine). It is important to remember that this binding is specific: T pairs with A, but not with C. The molecular recognition occurs because of the ability of bases to form specific hydrogen bonds: atoms align just right to make hydrogen bonds possible. Also note that a larger base (purine, A or G) always pairs with a smaller base (pyrimidine, C or T).

    Diagram showing the hydrogen bonds between nucleotides. Adenine is bound to thymine, and cytosine is bound to guanine.
    Figure 3. DNA chemical structure. Modification of DNA chemical structure by Madeleine Price Ball; CC-BY-SA-2.0

    Practice Questions

    True/False: DNA replication requires an enzyme.

    [practice-area rows=”1″][/practice-area]
    [reveal-answer q=”527189″]Show Answer[/reveal-answer]
    [hidden-answer a=”527189″]True. Most biological reactions rely on the enzyme to speed up the reaction. In the case of DNA replication, this enzyme is DNA polymerase.

    [/hidden-answer]

    What are the building blocks on DNA?

    1. Deoxyribonucleotides
    2. Fatty acids
    3. Ribonucleotides
    4. Amino acids

    [reveal-answer q=”93495″]Show Answer[/reveal-answer]
    [hidden-answer a=”93495″]Answer a. DNA is a double helix made up of two long chains of deoxyribonucleotides.

    [/hidden-answer]

    True/False: DNA replication requires energy.

    [practice-area rows=”1″][/practice-area]
    [reveal-answer q=”62103″]Show Answer[/reveal-answer]
    [hidden-answer a=”62103″]True. Making large molecules from small subunits (anabolism) requires energy. What supplies the energy? The building blocks themselves serve as a source of energy. As they get incorporated into the DNA polymer, two phosphate groups are broken off to release energy, some of which is used for making the polymer. Deoxyribonucleotides differ from nucleotides like ATP only by one missing oxygen atom.

    [/hidden-answer]

    We have the building blocks, a source of energy, and a catalyst. What’s missing? We need instruction about the order of nucleotides in the new polymer. Which molecule provides these instructions?

    1. Protein
    2. DNA
    3. Carbohydrate
    4. Lipid

    [reveal-answer q=”494506″]Show Answer[/reveal-answer]
    [hidden-answer a=”494506″]Answer b. We refer to this DNA as a template. The original information stored in the order of bases will direct the synthesis of the new DNA via base pairing.

    [/hidden-answer]

    There is one more thing required by the DNA polymerase. It cannot just start making a DNA copy of the template strand; it needs a short piece of DNA or RNA with a free hydroxyl group in the right place to attach the nucleotides to. (Remember that synthesis always occurs in one direction—new building blocks are added to the 3′ end.) This component starts the process by giving DNA polymerase something to bind to. What might you call this short piece of nucleic acid?

    1. A solvent
    2. A primer
    3. A converter
    4. A sealant

    [reveal-answer q=”529681″]Show Answer[/reveal-answer]
    [hidden-answer a=”529681″]Answer b. A primer is used to start this process by giving DNA polymerase something to bind the new nucleotide to.[/hidden-answer]

    Now that you understand the basics of DNA replication, we can add a bit of complexity. The two strands of DNA have to be temporarily separated from each other; this job is done by a special enzyme, helicase, that helps unwind and separate the DNA helices (Figure 4). Another issue is that the DNA polymerase only works in one direction along the strand (5′ to 3′), but the double-stranded DNA has two strands oriented in opposite directions. This problem is solved by synthesizing the two strands slightly differently: one new strand grows continuously, the other in bits and pieces. Short fragments of RNA are used as primers for the DNA polymerase.

    Diagram of both the leading and lagging strands the helicase splits the two strands and a DNA polymerase travels over both strands to create complementary strands.
    Figure 4. By Mariana Ruiz (DNA replication) Public Domain

    Practice Questions

    Which of these separates the two complementary strands of DNA?

    1. DNA polymerase
    2. helicase
    3. RNA primer
    4. single-strand binding protein

    [reveal-answer q=”197431″]Show Answer[/reveal-answer]
    [hidden-answer a=”197431″]Answer b. Helicase breaks the hydrogen bonds holding together the two strands of DNA.

    [/hidden-answer]

    Which of these attaches complementary bases to the template strand?

    1. DNA polymerase
    2. helicase
    3. RNA primer
    4. single-strand binding protein

    [reveal-answer q=”381621″]Show Answer[/reveal-answer]
    [hidden-answer a=”381621″]Answer a. DNA polymerase builds the new strand of DNA.

    [/hidden-answer]

    Which of these is later replaced with DNA bases?

    1. DNA polymerase
    2. helicase
    3. RNA primer
    4. single-strand binding protein

    [reveal-answer q=”2143″]Show Answers[/reveal-answer]
    [hidden-answer a=”2143″]Answer c. the RNA primer is replaced with DNA nucleotides.[/hidden-answer]

    In Summary: Major Enzymes

    Replication in eukaryotes starts at multiple origins of replication. A primer is required to initiate synthesis, which is then extended by DNA polymerase as it adds nucleotides one by one to the growing chain. The leading strand is synthesized continuously, whereas the lagging strand is synthesized in short stretches called Okazaki fragments. The RNA primers are replaced with DNA nucleotides; the DNA remains one continuous strand by linking the DNA fragments with DNA ligase. Below is a summary table of the major enzymes addressed in this reading, listed in rough order of activity during replication.

    Important Enzymes in DNA Replication
    Enzyme Function
    Topoisomerase Relaxes the super-coiled DNA
    DNA helicase Unwinds the double helix at the replication fork
    Primase Provides the starting point for DNA polymerase to begin synthesis of the new strand
    DNA polymerase Synthesizes the new DNA strand; also proofreads and corrects some errors
    DNA ligase Re-joins the two DNA strands into a double helix and joins Okazaki fragments of the lagging strand

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