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DNA: Discovery and Replication

By the turn of the century it was clear that the mysterious essence from males and females that combined in the embryo and determined the inherited traits of their offspring was not something otherworldly but rather a physical substance. In fact, with the help of a microscope, you could actually see it. Just watch a cell in mitosis. You can see the chromosomes, the carriers of heredity, segregate to each cell, bringing with them the genotype of the daughter cells. With the discovery that the genetic material was in the chromosomes came the realization that it might be possible to discover the chemical nature of the genetic material just as it had been possible to discover the chemical nature of many cell components. In other words, it might be possible to purify a gene. To do so you needed two things: 1) a method of isolating cell components, and 2) an assay for genetic materialness. As it happens the first problem, isolating cell components had already been solved in 1868 by Friedrich Miescher. He figured out a way to isolate the nuclei from cells found in pus and found that the main constituent of the nucleus was a compound he called nuclein. Nuclein turned out to be DNA. Thus nuclein became a candidate for being the genetic material but not the only candidate. After all, chromatin contains lots of protein too. Deciding which was the genetic material required an assay. This was a trickier problem. Ideally you would like to be able to take the candidate genetic material (protein or DNA or carbohydrate or whatever you think might be the genetic material) from, for instance, a round pea and and give it to a wrinkled pea plant and show that the wrinkled pea now became round. But you can't expect to just dip the wrinkled pea in round pea genetic material and expect it to become a round pea, can you? No, generally speaking, you cant. But in specific cases the answer is, amazingly, yes. Frederick Griffith was interested in developing a vaccine to Streptococcus pneumoniae so he spent a lot of time injecting live or heat-killed S. pneumoniae bugs into mice. He could culture the bugs on a petri plate too and worked on two strains S (smooth) and R (rough). In addition to having smooth and rough colonies on petri plates, the smooth bugs were virulent, they killed the mice and the rough bugs were non-virulent, the mice killed them. Of course heat killed S bugs were dead so they did not kill the mice. For some reason Griffith decided it would be a good idea to inject heat-killed S-bugs along with live R-bugs. This killed the mice dead, just as if he injected live Sbugs. In fact, when he looked in the dead mice, that's what he found, live S-bugs. The R-bugs had been transformed into S-bugs.

The substance from the S-bugs that transformed the R-bugs was called the transforming principle. Since properties of the S strain that were moved to the R strain were heritable, one might easily conclude that transforming principle = genetic material. Now, we have a simple assay for genetic materialness, i.e. the ability to transform R bugs into S bugs. Avery, Macleod and McCarty went to work to see what the transforming principle was. They found that no component of S bacteria could transform R bacteria except DNA. DNA was necessary and sufficient to transform. However, many people still needed convincing because they thought perhaps that the heredity that bacteria displayed was some weird bacteria thing that wasn't normal heredity or maybe the DNA wasn't really pure. So Al Hershey and Martha Chase did the following experiment. They took a virus that consists of a DNA core surrounded by a protein coat and radioactively labelled the DNA with 32P and or the protein with 35S. They allowed the virus to infect bacteria and then threw the mix in a blender. This had the effect that the protein coat was sheared off the bacteria, the idea being that whatever stayed behind with the bacteria must be the genetic material of the virus. Since the infected bacteria now produced more virus which contained 32P but almost no 35 S, it followed that the 32P-containing DNA must be the viral genetic material.

OK, so it's DNA. But that didn't explain how DNA could replicate itself and it didn't explain how DNA determined an organism's phenotype. Watson and Crick decided (as did many others) that the key was to get a better understanding of the three-dimensional structure of the DNA. Thus began one of the great scientific detective stories. They had a number of important clues that were widely known and were fortunate to have the unpublished data of Rosalind Franklin and Maurice Wilkins. First, enough organic chemistry had been done that people knew the chemical composition of DNA. It's a polymer of 4 subunits, the four nucleotides deoxyadenosine, deoxyguanosine, deoxycytidine and thymidine.

It was clear from the way that the sugars were linked that the polymer had a polarity defined by which end of its sugar backbone is sticking out, the 3' OH group or the 5' phosphate. It was commonly thought that the four occurred in equal proportions. However, Erwin Chargaff showed that the amount of A = T and the amount of G = C even though the ratio A+T/G+C can vary widely from one organism to the next.

Finally, Franklin and Wilkins had used the technique of X-ray diffraction to show that the DNA was doublestranded and helical. Putting these clues together, Watson and Crick came up with a model wherein the nitrogenous bases are in the middle and the phosphate backbone runs along the outside.

The charged phosphate backbone is exposed to the water. The bases, which are hydrophobic planar molecules, lie stacked on top of each other in the center. Most importantly the A from one strand always pairs with the T from the other strand and the G from one with the C from the other. This is important because: 1) the hydrogen bonds between A/T and G/C are the most stable and 2) by always pairing a purine and a pyrimidine, the distance between the two strands

remains constant (i.e. no bulging).

Finally, although each individual strand of DNA has a polarity, the two strands run in opposite directions, they are antiparallel. Because of base pairing, the sequence on one strand determines the sequence on the other strand. However, the sequence on the other strand is the complement of the 1st strand and runs in reverse. Therefore we say the strands are reverse compliments of each other.

The key here is that the structure immediately suggests two things: 1) The sequence of the nucleotides does not affect the overall structure so information can be encoded arbitrarily by the sequence of base pairs, and 2) the two strands bind by complementary base pairing so that the two strands contain identical information. If you separate the two strands and make a new partner strand for each, you create two identical DNA helices. You replicate the DNA. One question that arose was what happened to the two DNA strands during replication. There were three possibilities proposed: 1) conservative replication - they each made a new strand and the two old strands re-annealed (re-bound to each other) and the two new strands annealed, 2) semiconservative replication - each old strand made and remained annealed to a new strand,

and 3) dispersive replication - DNA would break apart and rejoin to produce four strands, each with a mixture of old and new DNA.

Meselson and Stahl showed that it was semiconservative. They were able to label the DNA by growing bacteria with a heavier isotope of nitrogen, 15N, making the DNA heavy. They could measure how heavy the DNA was by centrifugation in cesium chloride. After one duplication in the presence of normal nitrogen the duplicated DNA was half as heavy and after 2 duplications half the DNA was half as heavy and half the DNA was normal (light) weight. Only semiconservative replication would produce this result

So how does DNA duplication actually occur? It's pretty easy to synthesize a new DNA strand in a test tube. In fact, you can do it with some template DNA, the four nucleotides (in a triphosphate form) and a single enzyme. And one more critical thing, you need a ragged end. I mean by this a stretch of the DNA that is partly double stranded and partly single stranded as, for example, where one of the strands has been broken and half of it stripped away. The enzyme DNA polymerase, grabs a nucleotide that is complementary to the next nucleotide in line on the template strand. It takes the nucleotide and breaks the bond between the alpha phosphate (the one attached to the sugar) and the beta phosphate. It then attaches the alpha phosphate to the 3' hydroxyl group of the last nucleotide on the strand that's being extended.

Thus we say the DNA synthesis always occurs 5' to 3'. In the test tube the template DNA you start with is usually purified from some unfortunate organism and the DNA gets damaged in the process so it's always in pieces and has a many ragged ends. However, in a live cell this is not the case. A live cell has two problems: 1) it has to unwind the strands, and 2) it has to create a ragged end. So cells have two enzymes: helicase which expends energy to unwind the DNA, and primase which adds a short stretch of complementary RNA, a primer, which acts as a ragged end so the DNA polymerase (DNA polymerase III) can go to work. They don't initiate replication at random but at specific spots called origins of replication. Bacteria have one, eukaryotes have many along the length of the chromosome. As the helicases unwind the DNA in each direction from the origin of replication, a bubble forms with so-called replication forks on either end where the old double stranded DNA is being split to act as the template for the formation of two new strands.

Now if the cell were patient, it could wait for the strand from one origin to meet up with the primer from the next origin in line (in a circular bacterial DNA it would eventually run into its own primer at its own origin). The cell is not that patient. In order to deal with the single stranded DNA behind the primer in a replication bubble, the so-called lagging strand (as opposed to the leading strand), it keeps putting down primers every few hundred bases so that polymerase can be making the complementary strand for the lagging strand . Of course you end up with lots of short stretches of DNA beginning with a primer and running into the next primer along the the DNA. These stretches of DNA on the lagging strand are called Okazaki fragments. Finally, another polymerase, DNA polymerase I, chews up the RNA primers and, using the newly synthesized DNA as a primer, fills in the gaps. Then DNA ligase joins the ends of the newly synthesized strand and DNA replication is finished.

Almost. The polymerases make mistakes. The cell deals with this several ways: 1) The polymerase has a proofreading mechanism that immediately removes bases that are not complementary to the template strand. 2) 3) There is mechanism at work during recombination called mismatch repair. There is a mechanism at work the rest of the time called excision repair.

Excision repair also works on thymine dimers formed by exposure to UV light.