12 DNA: The Carrier of Genetic Information

Lecture Outline

I. Evidence that DNA is the hereditary material was first found in microorganisms

A. Early biologists believed that the genetic material was a protein

B. Frederick Griffith in 1928 studied pneumococcus bacteria in mice

1. Smooth (S) strain was virulent

2. Rough (R) strain was not virulent

3. Heat-killed S strain was not virulent (= avirulent)

4. A combination of heat-killed S strain and R strain was virulent

a) Posed the question: A Why?@

5. Something caused the R strain to become virulent (called it transformation)

C. This A transforming principle@ was later identified by Avery and colleagues in 1944 as DNA, while ruling out that it was from the lipid, protein, or polysaccharide fraction from the cells

D. In 1952, Alfred Hershey and Martha Chase= s experiments on bacteriophages ruled out that it was from the lipid, protein, or carbohydrate fraction from the cells

1. Bacteriophages are viruses that infect bacteria, and they are known to leave their protein coat (capsid) on the outside of the bacterium that they infect

2. Labeled the protein coat with radioactive sulfur, the DNA with radioactive phosphorus

3. Found that only the phosphorus was in the bacterium after infection with the bacteriophages, thus providing further evidence that DNA is the genetic material (at least in those viruses in this study)

4. Edwin Chargaff determined that the ratio of adenine to thymine and the ratio of guanine to cytosine was always the same in all organisms tested

II. Elucidated by Crick and Watson in 1953, the structure of DNA allows it to carry information and to be faithfully duplicated

A. Nucleotides can be covalently linked in any order to form long polymers

1. DNA is a nucleotide-based molecule containing deoxyribose, phosphate, and one of the 4 nitrogen-containing bases

2. The bases of DNA are purines (adenine and guanine) and pyrimidines (thymine and cytosine)

3. Nucleotides are covalently bonded with a sugar-phosphate backbone

a) The linkage forms a 3', 5' phosphodiester linkage

b) No matter the length, one end of the molecule has a free 5' carbon attached to a phosphate; the other end has a free 3' carbon attached to the hydroxyl group

B. DNA is made of two polynucleotide chains intertwined to form a double helix

1. Rosalind Franklin and Maurice Wilkins did x-ray diffraction analyses on DNA; this data was available to Crick and Watson

 

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a) Results showed that the molecule was helical and had several repetitive elements at 0.34 nm, 3.4 nm, and 2.0 nm apart

b) They deduced that the relatively flat base molecules were stacked like the rungs of a ladder

2. James Watson and Francis Crick deduced the correct structure of DNA

a) The molecule is a double helix of antiparallel strands (3' to 5' on one side, 5' to 3' on the other side) made up of a backbone of sugars and phosphates

b) The 0.34 nm repeat is due to the distance between the bases

c) The 3.4 nm repeat is due to the distance between a complete A turn@ of the helix every 10 base pairs in length

d) The 2.0 nm represents the width of the molecule

C. In double-stranded DNA, hydrogen bonds form between adenine and thymine and between guanine and cytosine

1. Pyrimidines are single-ringed molecules; purines have double-rings

a) Two hydrogen bonds form between thymine and adenine

b) Three hydrogen bonds form between cytosine and guanine

2. The bonding of bases is complementary; the sequence in one chain dictates the sequence in the opposite chain

3. This fits with data from Chargaff, as well as mathematical data from the x-ray diffraction studies, and ultimately became known as Chargaff= s rule

III. DNA replication is semiconservative

A. The structure of DNA clearly indicated the copying mechanism

1. A It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material@ (classic quote from Watson and Crick Nature article)

2. Using E. coli, Matthew Meselson and Franklin Stahl (1958) disproved the models of dispersive replication and conservative replication and showed strong evidence supporting semiconservative replication

a) Grew the bacteria in medium with radioactive 15N

b) Transferred to medium with 14N (nonradioactive)

c) Isolated the DNA by density gradient centrifugation and found that some of the DNA had only 15N, some had only 14N, and some had both, supporting semiconservative replication

d) After two cycles of replication, both incorrect models were disproved

B. Semiconservative replication explains the perpetuation of mutations

1. Mutations were known to arise in genes and transmitted to succeeding cells

2. The copying mechanism showed how novel base sequences would be passed to daughter molecules

C. DNA replication is complex and has a number of unique features

1. A great number of proteins and enzymes work together in replication; this treatment is greatly simplified (many genes are involved in this process)

2. DNA strands must be unwound during replication

 

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a) DNA helicase enzymes unwind the strands

b) Single-stranded binding proteins (SSBs), or helix destabilizing proteins, prevent immediate reformation of the double helix

c) Topoisomerases break and then rejoin the strands, A untying@ the knots that form

3. DNA synthesis always proceeds in a 5'± 3' direction

a) DNA polymerases can add only at the 3' end, hence growth is in the 5' to the 3' direction

b) Nucleotides become polymerized, and two phosphates are removed in the process

4. DNA synthesis requires an RNA primer

a) The RNA primer is synthesized at the point of initiation of replication

5. DNA replication is discontinuous in one strand and continuous in the other in the long DNA molecules of eukaryotic chromosomes

a) Replication begins at Y-shaped structures called replication forks

b) Synthesis of the leading strand is continuous; nucleotides are added at the 3' end of the new strand, so it is growing toward the fork

c) The lagging strand is synthesized in pieces called Okazaki fragments but is also added to the 3' end of the new strand, so it is growing away from the replication fork

(1) The same DNA polymerase molecules acts in both

d) Okazaki fragments are between 100 and 2000 nucleotides in length, and each is initiated by a separate RNA primer

e) Okazaki fragments are joined together by DNA ligase, which links the 3' hydroxyl of one fragment to the 5' phosphate next to it, forming a phosphodiester linkage

6. Most DNA synthesis is bidirectional

a) In prokaryotes, the circular DNA is opened up and synthesis occurs in both directions

b) In eukaryotes, when the double stranded DNA molecule separates, two replication forks form and the molecule replicates in both directions; this speeds replication

c) The linear DNA has many replication forks

7. The ends of eukaryotic chromosomes, the telomeres, present problems in replication

a) Telomeres have non-protein coding genes that repeat many times

b) Telomeres are not replicated completely after each S phase because of the RNA primer involvement

c) Ultimately, the entire telomere could be gone, and essential genetic material could be lost

d) Telomerase, discovered in 1984, is present in ever-dividing cells, including unicellular organisms, cancer cells, and germ-line cells

e) Shortening of telomeres contributes to cell aging and apoptosis

(1) Apoptosis was first experimentally described in the 1960s by Hayflick

f) Current research continues to focus on telomeres, telomerase, and medical applications such as anticancer and antiaging strategies, and the use of stem cells

 

 

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