Supplemental Lecture (99/02/22 update) by Stephen T. Abedon (abedon.1@osu.edu)

  1. Chapter title: DNA Replication
    1. A list of vocabulary words is found toward the end of this document
    2. Nucleic acid polymer replication is central to cell replication, organism replication, mutation, evolution, and life. It is also the most basic process of metabolism, one common to all organisms, cellular as well as non-cellular (i.e., viruses). In short, if anything in life truly has some purpose, then the prominant purpose of all of the processes of life is the replication of the molecules of heredity, DNA or genomic RNA, and the passage of these replicated molecules from parent to offspring.
    3. In this lecture we discuss the molecular mechanisms associated with DNA replication. In particular, we will consider the two main players in DNA replication: the DNA template and the whole series of of enzymes responsible for both manipulating the double helix and building new complimentary strands.
  2. DNA replication
    1. Sequence complementarity:
      1. The replication of DNA is built upon the existence of sequence complementarity.
      2. That is, within a double helix, one DNA molecule is made up of a sequence of nucleotides which is exactly complementary to its adjacent molecule.
      3. In fact, this complementarity, which is via hydrogen bonding between nitrogenous bases, is responsible for holding together the two molecules which make up the double helix and results in the sequence of one molecule exactly (though oppositely--i.e., complementarily) specifying the sequence of the molecule to which it is hydrogen bonded.
    2. Templating complimentary strands:
      1. Starting with one molecule of DNA it is possible to synthesize a second, complementary molecule of DNA by using the first molecule as a template.
      2. Since DNA normally exists as two complementary molecules in a double helix, using both molecules to template two new molecules results in the conversion of a single double helix into two.
      3. This template guided synthesis is DNA replication.
  3. Semiconservative DNA replication
    1. New + old strand:
      1. The process described in outline above is known as semiconservative DNA replication. This is as opposed to conservative DNA replication.
      2. In semiconservative DNA replication the original double helix is broken apart, one DNA molecule finding its way into one daughter double helix and the other, complementary, molecule finding its way into the other daughter double helix. See illustration below.
    2. A conservative process, on the other hand, would be one in which the parent double helix remained intact with a single daughter double helix formed upon DNA replication, one consisting entirely of newly synthesized DNA molecules. This latter process is actually the more complicated (i.e., requiring more steps to accomplish) and turns out to not represent how DNA replicates.
  4. Replication fork
    1. Rather than one molecule at a time, both DNA molecules making up a double helix are replicated simultaneously (or, more precisely, in parallel).
    2. To do this the two molecules are separated by a specific protein to turn the single double stranded DNA of the double helix progressively into two separated, single stranded DNA molecules.
    3. These single stranded DNA molecules are then employed to template the synthesis of daughter DNA molecules.
    4. Thus, the double helix is opened up into single stranded DNA in order to effect DNA replication, and the point at which both of these processes occur is called a replication fork.
    5. See illustration below.
  5. DNA polymerase
    1. DNA replication is more accurately described as polymerization. The enzyme which catalyzes this polymerization is called, not surprisingly, DNA polymerase.
    2. 5' to 3' polymerization:
      1. Note that DNA polymerases build DNA polymers by adding nucleotidess to the 3' end of DNA molecules (thus chains grow in the 5' to 3' direction and only in the 5' to 3' direction).
      2. In other words, the 5' phosphate is carried by the incoming nucleotide, rather than the 3' carbon of the last incorporated nucleotide.
    3. Lagging strand paradox:
      1. So what? When discussing the replication fork above we noted that DNA replication occurs as the double helix is opened up. However, one of the newly formed DNAs must be synthesized in a 3' to 5' direction (since there are only two possible directions of synthesis and the two daughter strands are synthesized in opposite directions due to the antiparallel arrangement of the double helix).
      2. But if there exist no DNA polymerases capable of polymerizing DNA in the 3' to 5' direction, how could this be?
  6. Discontinuous synthesis [Okazaki fragments, lagging strand, leading strand, DNA ligase]
    1. Synthesis of fragments:
      1. The answer to the above posed question (How DNA might be polymerized in the 3' to 5' direction if there exists no DNA polymerase that is capable of polymerizing DNA in the 3' to 5' direction?) is that it isn't.
      2. Instead, synthesis in the 3' to 5' direction is effected by delaying such synthesis until a reasonably large amount of single stranded DNA is present, then to synthesize that daughter DNA in the 5' to 3' direction. Fragments of DNA result from this discontinuous method of DNA replication (i.e., synthesis occurs towards the existing daughter DNA and upon the inevitable collision with this strand the DNA polymerase is neither able to continue polymerizing nor stitch together the two adjacent DNA molecules).
    2. Leading and lagging strands:
      1. We refer to this delayed, fragmented, daughter DNA as the lagging strand.
      2. This contrasts with the not fragmented, continuously synthesized DNA found on the other side of the replication fork, which is known as the leading strand.
    3. The fragments themselves are referred to as Okazaki fragments.
    4. DNA ligase:
      1. The enzyme which stitches them together into a single, unfragmented daughter molecule is called DNA ligase.
      2. See illustration_replication_forkillustration below.
  7. Illustration, replication fork
  8. Tape marking analogy
    1. Imagine you are unrolling white tape and coloring it black with a marker before the tape is rolled up onto a second roll. The tape is a special tape which has barbs that catch the marker if you try to color it in one direction, but not if you color it pushing the marker in the other direction.
    2. Coloring strategy depends on direction of barbs:
      1. You have two rolls of this tape. With one roll you can color as you unroll by keeping the marker directly and always in contact with the tape. The barbs are arranged on this tape such that the marker is not caught when used this way. The other roll, however, has the barbs arranged in the opposite direction. If you try to color this second roll as you unroll it, the marker will only become caught in a barb.
      2. Instead you enlist the strategy of pulling out a few centimeters of tape, then coloring it by pushing the marker in the opposite direction (i.e., from the roll toward the loose end, i.e., from the portion colored previously; see illustration below).
    3. This business of pulling out a bit and then coloring it "backward" is analogous to how DNA synthesis is achieved via discontinuous synthesis on the lagging strand (see illustration_replication_forkillustration below). The color as you go roll (the first discussed) is, on the other hand, colored in a manner analogous to that employed on the leading strand.
  9. Illustration, tape marking analogy
  10. Priming DNA polymerization
    1. Requirement for 3' primer:
      1. Not only are DNA polymerases incapable of polymerizing DNA in the 3' to 5' direction, but they are also incapable of initiating DNA polymerization in the absence of an existing 3' -OH (this has something to do with the DNA polymerases being optimized toward high fidelity sequence duplication---mutation avoidance---rather than high versatility).
      2. This is particularly a problem for the lagging DNA strand since synthesis via Okazaki fragment formation requires repeated initiation of DNA replication de novo.
    2. RNA priming:
      1. However, this paradox is neatly avoided by employing low fidelity RNA polymerases to prime DNA synthesis with RNA.
      2. Complementary RNA is polymerized in much the same manner as complementary DNA is later polymerized.
      3. This RNA supplies the necessary 3' -OH group to prime DNA polymerization. DNA polymerase then takes over from the RNA polymerase to effect DNA replication.
      4. The priming RNA is later clipped off and replaced with DNA by the DNA polymerase.
  11. Vocabulary
    1. Discontinuous synthesis
    2. DNA polymerase
    3. DNA replication
    4. Lagging strand
    5. Leading strand
    6. Priming DNA replication
    7. Replication fork
    8. Replication fork, illustration
    9. Semiconservative DNA replication
    10. Tape marking analogy, illustration
  12. Practice questions
    1. What is the reason RNA priming is necessary to initiate DNA replication? (<20 word answer) [PEEK]
    2. Why, during DNA replication, does the lagging strand "lag?" (<20 word answer) [PEEK]
    3. Describe the end product of semiconcervative DNA replication in such a way that convinces me that you know the difference between it and a hypothetical product of conservative DNA replication. [PEEK]
    4. _________ is one correct reason why Okazaki fragments are created during lagging strand DNA synthesis. (circle one correct answer) [PEEK]
      1. DNA is only polymerized in the 3' to 5' direction.
      2. DNA polymerase requires RNA priming.
      3. DNA is only polymerized in the 5' to 3' direction.
      4. DNA polymerase requires a 5' -0H on the growing polymer.
      5. leading strand synthesis is continuous.
      6. incoming nucleotides have a phosphate group attached to their 3' OH.
  13. Practice question answers
    1. DNA polymerase requires a 3' -OH, RNA polymerase does not.
    2. any number of answers including: discontinuous replication, Okazaki fragments, DNA polymerase can only synthesize in the 5' to 3' direction . . .
    3. DNA molecule where one strand is parental DNA and the other strand is the daughter DNA product of DNA polymerization.
    4. iii, DNA is only polymerized in the 5' to 3' direction.
  14. References
    1. Raven, P.H., Johnson, G.B. (1995). Biology (updated version). Third Edition. Wm. C. Brown publishers, Dubuque, Iowa. pp. 280-299.