Reading Assignment - Mader: Chapter 13|
Scientists had developed a number of criteria that had to be met for a molecule to be the genetic material.
- it should be able to store information within its structure; this information must include the ability to control development and all metabolic activities of the cell
- it must be stable enough so that it can be accurately copied and transmitted for many generations; and
- it must be able to undergo spontaneous change (mutation) so that it can provide the genetic variability required for evolution
- Discovery of DNA Function
- In 1868-9, Friedrich Miescher first isolated DNA from pus and fish sperm; called “nuclein” which was rich in phosphorus but lacked sulfur
- Other researchers discovered that nuclein contained an organic acid – nucleic acid. Two types were soon discovered: DNA (deoxyribonucleic acid) and RNA (ribonucleic acid)
B. A Puzzling Transformation
- In 1928-31, Griffith studied Streptococcus pneumoniae, which causes a form of
bacterial pneumonia in mammals
- Strain S produced smooth colonies and was pathogenic; the smoothness was due to the presence of a capsule on the outside of the bacterium; the capsule is genetically determined
- Strain R produced rough colonies and was not pathogenic
- Heat killed colonies of strain S were not pathogenic
- Live R + heat killed S were pathogenic
- Conclusion: Genetic material from the dead S transformed the living R bacteria into a pathogenic form
- In 1944, Avery and his coworkers used enzymes to demonstrate that the transforming factor was DNA. But at this time most biologists believed that genes were made of proteins and few took note of this work.
C. Bacteriophage Studies
- Delbruck, Hershey, and Luria studied viruses that infect bacteria and are called bacteriophages
- Viruses adsorb (attach to the surface) to a host bacterial cell, inject their contents into the cell, and the host cell begins to produce viral proteins, viral nucleic acids and to assemble these into new viruses.
- In 1952, Hershey and Chase
conducted experiments to confirm Avery's conclusions that DNA was the transforming factor
- By first growing bacteriophages in radioactive 35S or 32P, it was demonstrated that DNA, not protein, enters the host cell
These experiments convinced biologists that heritable traits are encoded in DNA
- DNA Structure
A. Components of DNA
- Long before 1952, it was known that DNA is a polymer of nucleic acid built from four kinds of nucleotides
- A nucleotide consists of a five-carbon sugar, a phosphate group and a nitrogen containing base
- Each nitrogen base is either adenine (A), guanine (G), thymine (T), or cytosine (C)
- By 1949, Chargaff demonstrated that:
- the four bases differ in relative amounts from one species to another
- in any DNA molecule, the amount of A=T, and the amount of G=C
- could the arrangements of the four bases represent the hereditary information?
Wilkins, Franklin and others studied DNA with X-ray diffraction
- this technique can reveal the position of groups of atoms in a molecule
- it revealed DNA to be long and thin, with a uniform diameter of 2 nanometers
- DNA also shows repeats of 0.32 nanometers and 3.4 nanometers
- DNA could be helical
B. Patterns of Base Pairing
- in the early 1950's Watson and Crick deduced the structure of DNA
- if DNA has a uniform diameter, then it could be a double helix with A pairing with T, and C with G
- each pair of nucleotides on opposite strands could be held together with hydrogen bonds, like the rungs of a ladder
- the DNA of different species show variation in the sequence of base pairs
- DNA Replication
- go to DNA Workshop Activity and do the DNA Replication Animation
A. How DNA is duplicated
- first, the two strands of DNA unwind and expose their base pairs; hydrogen bonds are broken by the enzyme helicase
- then, free (complementary) nucleotides pair with exposed bases
- thus, replication results in new DNA molecules that consist of one "old" strand and one "new" strand
- that is, each old strand of DNA serves as a template or model to form the new strand
- this method is called SEMICONSERVATIVE REPLICATION
B. A Closer Look at Replication
go through the notes and try the animation
- Origin and Direction of Replication
- replication begins at an origin of the DNA molecule as the double helix unwinds
- viral or bacterial DNA has a single origin while eukaryotic DNA may have several origins
- unwinding occurs in both directions away from the point of origin
- strand assembly occurs behind each fork as the double helix continues to unwind
- Energy and Enzymes for Replication
- unwinding requires many kinds of enzymes
- DNA POLYMERASES assemble the nucleotides into nucleic acids and "proofread" the new bases for mismatched pairs, which are then replaced with the correct base pairs
the energy to drive the replication is derived from splitting phosphates from triphosphates (ATP--->ADP)
Source of the following materials http://www.ncc.gmu.edu/dna/replicat.htm
How does the DNA polymerase enzyme know where to begin synthesis? Is there some sort of marker, a start point?
YES; the start point for DNA polymerase is a short segment of RNA known as an RNA primer.
The very term "primer" is indicative of its role which is to "prime" or start DNA synthesis at certain points. The primer is "laid down" complementary to the DNA template by an enzyme known as RNA polymerase or Primase.
The DNA polymerase (once it has reached its starting point as indicated by the primer) then adds nucleotides one by one in an exactly complementary manner, A to T and G to C.
How does the polymerase "know" which base to add?
DNA polymerase is described as being "template dependent" in that it will "read" the sequence of bases on the template strand and then "synthesize" the complementary strand. The template strand is ALWAYS read in the 3' to 5' direction (that is, starting from the 3' end of the template and reading the nucleotides in order toward the 5' end of the template).
The new DNA strand (since it is complementary) MUST BE SYNTHESIZED in the 5' to 3' direction (remember that both strands of a DNA molecule are described as being antiparallel). DNA polymerase catalyzes the formation of the hydrogen bonds between each arriving nucleotide and the nucleotides on the template strand.
In addition to catalyzing the formation of Hydrogen bonds between complementary bases on the template and newly synthesized strands, DNA polymerase also catalyzes the reaction between the 5' phosphate on an incoming nucleotide and the free 3' OH on the growing polynucleotide (what we know is called a phosphodiester bond!).
As a result, the new DNA strands can grow only in the 5' to 3' direction, and strand growth must begin at the 3' end of the template, right? Again, note that a phosphodiester bond is formed between the 3' OH group of the sugar and the 5' phosphate group of the incoming nucleotide.
Because the original DNA strands are complementary and run antiparallel, only one new strand can begin at the 3' end of the template DNA and grow continuously as the point of replication (the replication fork) moves along the template DNA. The other strand must grow in the opposite direction because it is complementary, not identical to the template strand.
The result of this side's discontiguous replication is the production of a series of short sections of new DNA called Okazaki fragments (after their discoverer, a Japanese researcher). To make sure that this new strand of short segments is made into a continuous strand, the sections are joined by the action of an enzyme called DNA ligase which LIGATES (zips up) the pieces together by forming the missing phosphodiester bonds!
It is important to realize that DNA polymerase can only work in the 5' to 3' directions. The reason behind this is because 3' is more stable than 5' when attaching a new nucleotides. If DNA polymerase ran in the other direction then there is the risk that the phosphate group could break off.
So when DNA polymerase is working it only goes in the 5' to 3' direction. However there are two sides, and one runs in the 5' to 3' direction, but the other runs from 3' to 5'. So while DNA polymerase can work continuously in the 5' to 3' direction it has to work in spurts on the 3' to 5' direction, which are called Okazaki fragments. The 5' to 3' is called the leading strand while the other is called the lagging strand.
The last step is for an enzyme to come along and remove the existing RNA primers (you don't want RNA in your DNA now that the primers have served their purpose, do you?) and then fill in the gaps with DNA. This is the job of yet another type of DNA polymerase which has the ability to chew up the primers (dismantle them) and replace them with the deoxynucleotides that make up DNA.
Since each new strand is complementary to its old template strand, two identical new copies of the DNA double helix are produced during replication. In each new helix, one strand is the old template and the other is newly synthesized, a result described by saying that the replication is
semi-conservative. Crick described the DNA replication process and the fitting together of two DNA strands as being like a hand in a glove. The hand and glove separate, a new hand forms inside the old glove, and a new glove forms around the old hand. As a result, two identical copies now exist.