DNA

1.         Evidence for DNA as genetic material

            a.         Griffith, 1928 (Fig. 15.1) - In his work with Streptococcus pneumoniae, Griffith realized that some “transforming” agent was exchanged between bacteria which enabled to acquire traits from one another. The use of heat to inactivate cells suggested that the agent was not protein. This phenomenon is now called transformation - a change in phenotype by taking genetic material from the environment.

            b.         Avery, et al., 1944 - This team isolated various chemicals from bacteria and used them to try transform bacteria. Only DNA worked.

            c.         Hershey and Chase, 1952 (Fig. 15.2) - Being essentially protein and nucleic acid, viruses were good candidates for studying this problem. They used radioactive S to label the protein of a bacteriophage and allowed it to infect the host bacterium. After centrifugation, bacterial cells would be in the pellet. They found the radioactive protein was found in the supernatant, showing that protein is not injected into the host. Then, radioactive P was used to label the nucleic acid of the virus. After infection and centrifugation, the radiation was in the pellet with the bacterial cells. This showed that nucleic acid is injected into host bacterium during infection. This provided additional evidence that DNA was the genetic material.

            d.         Chargaff, 1947 - He showed that in different species, DNA contained different amounts of the four nitrogen bases, suggesting that its composition was diverse. If the nucleotides were chosen randomly, species would be expected to have the same relative amounts of each nucleotide. He also realized that A = T and C = G, although he could not explain this observation.

2.         Structure of DNA (Fig. 15.3 and 15.5)

            a.         Wilkins and Franklin used X-ray diffraction to attempt to find the structure of DNA.

            b.         Watson and Crick (1953) did little original work but interpreted the X-ray data to elucidate the structure of DNA. They used the data collected by Franklin to build scale models of DNA.

            c.         The basic structure is two chains wound together in a spiral (i.e., a double helix).

            d.         The sides of the chains are made of alternating sugars and phosphates, like the sides of a ladder.

            e.         The rungs of the ladder are made of the four nitrogen bases paired AT and CG. This is called complementary base pairing. Why must A bond with T and C with G? The two strands are held together by hydrogen bonding between bases.

            f.         Note that the chains have direction - one end (5') has a free phosphate, the other (3') a free hydroxyl (-OH).

            g.         In the double stranded molecule, the two strands are upside down to one another. This arrangement is called antiparallel. (Fig. 15.11)

3.         Replication of DNA (Fig. 15.14) - This is the process by which DNA is doubled so that each daughter cell gets a copy during cell division. It is important for this process to be high fidelity.

            a.         Three models for replication had been proposed (Fig. 15.7). The correct model was determined by an experiment (Fig. 15.8) done by Meselson and Stahl (late 1950s). Bacteria growing for several generations on a heavy isotope of N (¹⁵N) were allowed to grow for one generation (i.e., DNA replicated once) on a light isotope of N (¹⁴N). This meant that any new (parent) DNA would be lighter than old (daughter) DNA. The cells had DNA of one weight, indicating that it was constructed from half heavy (old) and half light (new) N. After a second generation (i.e., another replication) the cells had DNA of two distinct weights. Half was all light and half was a mixture of light and heavy. This showed that replication is semi-conservative.

            b.         When DNA is copied, the two strands separate and each strand serves as a template for building a new, complementary strand. One at a time, nucleotides line up along the template strand according to base pairing rules.

            c.         Origin of Replication

                        i.         A specific sequence of nucleotides marks the origin or starting point.

                        ii.        Humans have hundreds of origins from which replication proceeds on both strands in both directions.

                        iii.       The area where the strands are separated to expose the bases is called the replication fork.

                        iv.       The enzyme helicase separates the strands.

            d.         Elongating a new strand

                        i.         After the strands are separated, DNA polymerase “reads” the exposed bases on the template strand and attaches new bases by complementary base pairing. Note that this process is decreasing entropy greatly so it must require energy. The energy comes from the substrates themselves which are nucleoside triphosphates (Fig. 15.10). The loss of two phosphates from the substrate provides the energy to drive the reaction.

                        ii.        DNA polymerase can only attach the 5' phosphate (P) of one nucleotide to the 3' hydroxyl (OH) of another nucleotide that is already part of a strand. The enzyme can only work by building a new strand in the 5' ➝ 3' direction.

            e.         Problem of antiparallel strands (Fig. 15.12)

                        i.         The C5 phosphate of one nucleotide is attached to the C3 hydroxyl of an adjacent nucleotide. Therefore, the strand has a free 3' OH at one end and a free 5' P at the other. Remember, the molecule is arranged with the strands going in opposite directions so the 3' end of one strand is aligned with the 5' end of the other.

                        ii.        DNA polymerase adds nucleotides only to the 3' end but can only do this on one strand, the leading strand.

                        iii.       The other strand has a 5' P at the end rather than the 3' OH DNA polymerase needs. This strand, the lagging strand, must be made in an overall 3' ➝ 5' direction. To do this, the new strand is made in short fragments, called Okazaki fragments, going in the opposite direction from the leading strand. Another enzyme, DNA ligase, then fills in the gaps to join the fragments together. Notice that although the strand overall is growing in a 3' ➝ 5' direction, the Okazaki fragments individually grow in a 5' ➝ 3' direction.

            f.         Priming DNA Synthesis (Fig. 15.13)

                        i.         Remember that DNA can only attach the 5' phosphate (P) of one nucleotide to the 3' hydroxyl (OH) of another nucleotide that is already part of a strand. This limitation means that the enzyme cannot begin synthesis de novo - it requires something to build on.

                        ii.        A primer is a short piece of RNA that is constructed on the template to serve as a starting point for DNA polymerase. The enzyme primase builds the primers, which are about 10 nucleotides long.

                        iii.       Later, the primers are replaced by DNA.

            g.         Error rate

                        i.         Complementary base pairing allows an error rate of 1/10,000 bp. Why do these errors occur?

                        ii.        DNA polymerase checks for these errors by checking the width of the helix and reduces the rate to 1/10⁸.

                        iii.       DNA is constantly exposed to chemicals, viruses, and radiation which cause damage. This damage is repaired by>50 known enzymes that constantly check DNA for errors. These combined efforts reduce the error rate to 1/10⁹.