Ch. 16DNA: The Genetic Material

I. IntroA. In 1953, James Watson and Francis Crick

presented their model of DNA to the world.B. Nucleic Acids (DNA: Deoxyribonucleic acid,

RNA: Ribonucleic acid) have a unique abilityto replicate itself.

DNA’s ability to replicate itself precisely is important for its transmission from one generation to the next.

II. The search for genetic material led to the discovery of DNA and its structure.

A. Before the 1940’s it was thought that proteins were the genetic material.

B. The genetic role of DNA was first researchedby Frederick Griffith in 1928.

1. Studied Streptococcus pneumoniae, a bacterium that causes pneumonia in mammals.a. He discovered one strain that was

nonvirulent (harmless) - R strain.b. Another strain was virulent (causes

pneumonia) – S strain.2. His experiment:

a. Mixed heat-killed S strain with live R strain and injected it into mice.

b. The mouse died and Griffith took a blood sample.

c. He found that some of the R strain had changed into the S strain. He called this transformation. Some chemical component had changed theR strain into S strain.

After Griffith’s experiment, researchers tried to discover this transforming material.

C. Finally in 1944, Oswald Avery, Maclyn McCarty and Colin MacLeod announced that the transforming substance was DNA.1. They took various chemicals from the

heat-killed pathogenic bacteria and triedto transform nonharmless bacteria withthem. Only DNA worked.

D. In 1952, Alfred Hershey and Martha Chase showed that DNA was the genetic material of the phage T2 (a virus that infects e. coli bacteria).

1. They studied bacteriophages – virusesthat infect bacteria. They knew that viruses need bacteria in order to replicate.

2. Since viruses have simple structure, theywanted to know whether it was their protein coat or DNA that was the geneticmaterial.

3. Their experiment:a. They had two batches of viruses:

-Viruses with radioactive sulfur (S-35) labeling their protein coat.

-Viruses with radioactive phosphorus (P-32) labeling their DNA.

b. They allowed for the two batches toinfect bacteria. After infection, theyput the virus/bacteria mixture in a blender so that the viral parts outsideof the bacteria could be separated.

c. They centrifuged the mixture so thatthe bacteria form a pellet at the bottom of the tube.

d. Then they tested the bacteria for radioactivity. Found radioactivity inside bacteria.

e. They concluded that the injected DNA,radioactively labeled was the geneticmaterial.

E. In 1947, Erwin Chargaff had developed a series of rules based on a survey of DNA composition in organisms.1. He already knew that DNA was a polymer

of nucleotides consisting of a nitrogenous base, deoxyribose, and a phosphate group.2. The bases could be adenine (A), thymine

(T), guanine (G), or cytosine (C).3. Chargaff noticed that the DNA

composition varied from species to species.

4. He found that the bases were present inall species in very regular ratios:-The number of Adenine = Thymine-The number of Cytosine = Guanine

F. Watson and Crick: By the 1950’s it was nowaccepted that DNA was the genetic material.The race was on to discover its structure.

-Linus Pauling-Maurice Wilkins and Rosalind Franklin

G. Wilkins and Franklin used X-Ray crystallo-graphy to study the structure of DNA.

1. From their pictureof DNA, Watson and Crick were ableto see its helicalstructure.

H. Double-Helix model of DNA proposed byWatson and Crick:1. DNA is made up of nucleotides.

2. DNA looks like a ladder. It has two strands, each strand with the sugar-phosphate chains on the outside and the nitrogenous bases on the inside.3. The nitrogenous bases paired up, forming

the rungs of the ladder. 4. The ladder is then twisted, forming a coil.

5. The nitrogenous bases are paired up veryspecifically:

A – TG - C

purines(double ring)

pyrimidines(single ring)

-Only a pyrimidine-purine pairing would produce the 2-nm diameter indicated by the X-ray data.

-The A & T, C & G form hydrogen bonds between one another:-A = T (two)-G = C (three)

** This confirms Chargaff’s observations.

The Structure of DNA

• http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/DNA_structure.html

6. The sequence of nucleotides on one DNAstrand can vary in numerous ways. Eachgene has a specific sequence of nucleotides.

A portion of gene has the following sequence of nucleotides:

ATGGACTTC

-T-A-C-C-T-G-A-A-G

-Watson and Crick presented their DNA model in 1953.-They, along with Maurice Wilkins won the Noble Prize in Medicine in 1962.

Watson Crick

III. DNA Replication:A. After Watson and Crick presented their DNA

model, they wrote about how DNA replicates.1. They said that the two strands of DNA

are complimentary to one another.2. When they are separated, they can act

as templates for synthesizing a new strand of DNA.

3. Watson and Crick’s model of replication was called “Semiconservative replication.”

-This means that when two strands of DNA are made, each one will have a new strand and an old one. The old strands will act as “templates” to the new complimentary strand.

B. Experiments done in the late 1950s by Matthew Meselson and Franklin Stahl supported the semiconservative model.1. In their experiments, they labeled the

nucleotides of the old strands with a heavy isotope of nitrogen (15N) while any new nucleotides would be indicated by a lighter isotope (14N).

2. After they labeled the DNA and let it replicate, they found that each DNAmolecule had one strand labeled with N-15 and the other with N-14.

3. They then allowed for the DNA to replicate once more and they found thatthe only strands with N-15 were the original two strands of DNA.

Meselson-Stahl Experiment

• DNA Replication

• http://www.sumanasinc.com/webcontent/anisamples/majorsbiology/meselson.html

C. More than a dozen enzymes and proteins carry out DNA replication:

1. E. coli can replicate its DNA in less thanan hour. Human cells can replicate its 6 billion base pairs in only a few hours.

Replication is highly accurate; only one error per billion nucleotides.

D. DNA replication starts at the origins of replication.

1. In bacteria, there are very specificnucleotide sequences that enzymes recognize as sites where replication begins.

2. In eukaryotes, there are many sites on the DNA strand where replication takesplace.

3. At the origin of replication, a replicationbubble forms, where new DNA strands are elongated in both directions.

4. DNA Polymerase is the enzyme that elongates the new DNA at a replication fork.

-The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells. -The nucleotides that are attached to the newly formed strands are called nucleoside triphosphates.Each has a nitrogen base, deoxyribose, and a triphosphate tail.

-As each nucleotide is added, the last two phosphate groups are hydrolyzed to form pyrophosphate.

-The exergonic hydrolysis of pyrophosphate to two inorganic phosphate molecules drives the polymerization of the nucleotide to the new strand.

E. The strands in the double helix are antiparallel.

1. The sugar-phosphate backbones run in opposite directions.

2. One strand goes from 3’ 5’

direction. The other strand goes from 5’ 3’ direction.

3. DNA polymerases can only add nucleotides to the free 3’ end of a growing DNA strand.

-DNA can only replicate in the 5’ 3’ direction.

-Leading strand replicates from 5’ 3’.-Lagging strand replicates from 5’ 3’, but by forming Okazaki fragments.-The Okazaki fragments (100-200 nucleotides), are then joined by DNA ligase.

F. DNA replication starts with a primer (a short fragment of RNA).

1. A primer iscreated by anenzyme calledprimase.

2. Once the primeris made, DNApolymerase canstart adding nucleotides at the3’ end.

3. The primer isthen converted

into deoxyribo-nucleotides.

4. Only one primer isneeded for the leading strand. A new primer is needed for eachOkazaki fragment.

G. Enzymes involved in DNA replication:1. Primase: 2. DNA Polymerase:

3. DNA Ligase:

Creates a primer.Adds nucleotides to the 3’ end; replacesRNA primer.

Joins the Okazaki fragments.

4. Helicase: Untwists DNA and separates the template strands at the replicationfork.

5. Single-strand binding proteins: keepthe template strands apart during replication.

H. Enzymes proofread DNA during replicationand repair existing damaged DNA.

1. Mistakes during DNA synthesis can occur at a rate of one error per 10,000 base pairs.

2. DNA Polymerase proofreads the newDNA strand. If there is a mistake, DNApolymerase removes the incorrect nucleotide and resumes synthesis.3. After proofreading, the error rate is

one per billion nucleotides.

I. Harmful chemicals, radioactive emissions, X-rays, and ultraviolet light can change nucleotides.

1. There are over 130 enzymes that helprepair damaged and mutated DNA.

Also, under normal cellularconditions, DNA can undergo spontaneousmutations.

2. Defects in enzymes that help repair mismatched nucleotides are associatedwith colon cancer.

3. Nucleases are enzymes that excise (cut out) damaged nucleotides. After they are cut out, the gap is filled in with the correct nucleotide via DNApolymerase and ligase.

Example: The inherited disorder called Xeroderma Pigmentosum causes an individual to be very sensitive to sunlight.UV light can cause two adjacent Thyminenucleotides to form a dimer. The dimer buckles the DNA strand and interferes withDNA replication. Causes skin cancer.

J. The End-Replication problem: When eukaryotic DNA replicates, a gap is left at the 5’ end of each new strand because DNApolymerase can only add nucleotides at the3’ end.

Gap formedwhere primer previouslyexisted.

1. To help this problem, eukaryotic DNAhave telomeres at their ends. Telomeresare not genes but the sequence

TTAGGG

repeated between 100 to 1,000 times.2. The telomeres prevent any important

genes from being deleted over time dueDNA shortening with repeated replication.3. The enzyme, telomerase, catalyzes the

lengthening of telomeres.

-Telomerases have a short RNA fragment that serves as a template for a new telomere.