Chapter 16: Molecular Basis of Inheritance

The Race for the Double Helix .

The Search for the Genetic Material

A. Friedrich Miescher (1868; Swiss)

nuclein

B. Robert Feulgen (1914; German) DNA staining

C. Frederick Griffith (1928; English)

Streptococcus pneumoniae

Injected mice with a virulent strain (smooth because of a coat)

Streptococcus pneumoniae

Injected mice with a nonvirulent strain (rough)

It lacks a capsule; is harmless

Streptococcus pneumoniae

Boiled the virulent strain.

Heat-killed S cells are harmless

Streptococcus pneumoniae

Boiled the virulent S strain.

Combined it with non-virulent R strain

What do you predict happened to the mice?

Figure 16.1 Transformation of bacteria

We know this process today as bacterial transformation.

But what is the transforming factor?

Is it DNA or Protein?

THE SEARCH FOR THE TRANSFORMING FACTORD. Avery, Macleod and McCarty

S strain fractionated

• RNA

• Protein

• Lipid

• Carbohydrate

• DNA

E. HERSHEY AND CHASEbacteriophages

Figure 16.2ax Phages

Figure 18.4 The lytic cycle of phage T4

Figure 16.2b The Hershey-Chase experiment

The experiment showed that T2 proteins remained outside the host cell during infection, while T2 DNA enters the cell

F. Hershey-Chase Experiment

• Results published in 1952

• 1969 Nobel Prize– Delbruck, Luria and Hershey

F. Other Evidence

1. Amount of DNA doubles prior to mitosis

2. Diploid chromosomes have twice as much DNA as haploid sets found in the gametes of the same organism.

G. Erwin Chargaff 1947

1. Studied the DNA of various species

2. While the % of A,T,C,G varied between species, the amount of A = T, and C = G

3. Chargaff’s Rules

Figure 16.3 The structure of a DNA stand

Figure 5.1 Building models to study the structure and function of macromolecules

Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA

Deductions about DNA made from Franklin’s Photo

1. DNA is a helix with a uniform width of 2 nm.

2. Purine and pyrimidine bases are stacked .34 nm apart

3. The helix makes one full turn every 3.4 nm along its length.

4. There are ten layers of nucleotide pairs in each turn of the helix.

Model postulates:

1.The sugar-phosphates alternated on the exterior of the molecule

2.The nitrogen bases were in the interior, each one bonded to a sugar.

Model postulates:

3. The two sugar-phosphate backbones of the helix are antiparallel.

4. A purine (adenine or guanine) must pair with a pyrimidine (cytosine or thymine).

5. This base pairing dictates which pairs of bases can hydrogen bond: A-T, C-G

6. Base pairing rule explains Chargaff’s rules

Figure 16.5 The double helix

Unnumbered Figure (page 292) Purine and pyridimine

Figure 16.6 Base pairing in DNA

Figure 16.8 Three alternative models of DNA replication

Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 2)

Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 3)

ONE Layers was obtained.

What does this show?

Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 4)

After 2nd replicationTWO bands appear. What does this show?

B. Replication Background

Just how big is your genome?

Your genome is 6B bp (3B X 2 chromosomes)

If printed out the size of your textbook font, this would fill

1200 Campbell Biology Texts!

B. Replication Background

1. 5M bp with E. coli; 3B bp in humans!

2. Complex

3. Rapid (up to 500 nucleotides/second in bacteria, 50/sec in human cells)

4. Accurate ( errors only 1/10B)

5. Enzymes: Requires the cooperation of over a dozen

C. Process of ReplicationOrigins of Replication: Bacteria have only 1

Replication forks

Eukaryotes may have hundreds or thousands of origins of replication

Incorporation of a nucleotide into a DNA strand

The nucleotides added are actually triphosphates; the hydrolysis of the pyrophosphate is the exergonic reaxthat drives polymerization.

Figure 16.12 The two strands of DNA are antiparallel

New nucleotides can only be added at the 3’ end. This presents a problem!

Figure 16.14 Priming DNA synthesis with RNA

But first . . .

Primers: short segments of RNA polymerized by RNA primase

Figure 16.14 Priming DNA synthesis with RNA

Then . . .

DNA polymerase III

•13 known active sites

•Self-correction unit (limits errors to 1/107

bp)

•Can only add new bp to 3’ end

DNA Synthesis• All synthesis is done from 3’ to 5’ end.

• Proceeds smoothly in one direction = LEADING STRAND

• Discontinuous in the other direction =LAGGING STRANDS

• OKAZAKI FRAGMENTS

Synthesis of leading and lagging strands during DNA replication

LIGASE

Is the “glue” required to connect the fragments

A summary of DNA replication

Enzymes of SynthesisHelicase

Untwists

Topoisomerase

Nicks

SSB

Holds apart

Enzymes of SynthesisPrimase

Makes RNA Primer

Ligase

Connects Okazaki fragments

DNA Polymerase III

Adds new nucleotides 5’ to 3’

DNA Polymerase I

Removes RNA primer; replaces it with DNA

A--CATALYZES 5’-3’ ADDITON OF NUCLEOTIDES

E- PROOFREADS 3’-5’

B2 DIMER CLAMPS AROUND DOUBLE HELIX (yellow and blue)

Figure 16.15 The main proteins of DNA replication and their functions

Nobel Prize 1959

Kornberg for DNA Polymerase

Nobel Prize 1962

Watson, Crick and Wilkins

PROOFREADING AND REPAIR

Amnesty Order of Caesar:

EXECUTE NOT, LIBERATE!

However, slightly altered:

EXECUTE, NOT LIBERATE!

PROOFREADING AND REPAIR

DNA is only macromolecule to be repaired

a. Correct nucleotide has higher affinity for moving polymerase

b. DNA polymerase is a “self-correcting” enzyme; has a 3’ to 5’ proofreading exonuclease

PROOFREADING AND REPAIR

Errors in completed molecule: 1/1 billion

c. Mismatch repair enzymes; mutation in one of these assoc. with colon cancer.

d. Reactive chemicals and UV can contribute to DNA alterations.

e. There are over 50 130 repair enzymes in humans!

A. Causes of DNA Damage

Environmental Agents

a. UV Light

1. Thymine or cytosine dimers

2. Distortion interferes with replication and protein synthesis

Figure 16.17 Nucleotide excision repair of DNA damage

A. Causes of DNA Damage

Environmental Agents

b. Ionizing Radiation: X rays, Atom bomb, 1986 Chernobyl

1. Radiation reacts w/ DNA or water molecules

A. Causes of DNA Damage

Environmental Agents

c. Chemical Agents (Carcinogens)

1. Benzopyrene

2. Cigarette smoke, auto exhaust

3. Dioxin

PROOFREADING AND REPAIR

Nucleotide Excision Repair:

• DNA Polymerase I (a nuclease) snips out wrong base, puts in correct nucleotide

• DNA ligase seals it back

• Thymine dimers are common with UV damage; Xeroderma pigmentosum

B. DNA Repair: Enzymatic Processes

1. Selection of Correct Nucleotide

a. Correct base is most energetically favorable

b. Error occurs about every 1/100,000 bases

B. DNA Repair: Enzymatic Processes

2. “Proofreading”

a. Each nucleotide must be complementary

b. Exonucleases remove mismatched nucleotides as added

c. Now errors reduced to 1/10M bp

B. DNA Repair: Enzymatic Processes

3. Mismatch Repair

a. Occurs after synthesis

b. Proteins excise damage

c. Polymerases synthesize new strands

d. Error rate reduced to 1/10B bp

C. REPAIR ENZYMES1. Often function after damage has been

done

2. Over 130 known in humans

a. Photolyase: activated by absorbing visible light; breaks dimers apart

b. UV repair enzymes: uvrA, B, C

These are nucleases: Remove damaged sections of nucleotides

C. REPAIR ENZYMES

4. CancerOften caused by unrepaired DNA

damage

Ex. Xeroderma pigmentosum

Xeroderma pigmentosum

1. Skin cells defective in excision repair enzymes

2. Can develop hundreds of skin cancers

3. “freckled”

4. Internal Cancers

TELOMERES Revisited

1. 1972: James Watson noticed that the DNA polymerases could not start at the very tip of a DNA strand.

2. Analogy: Copy Machine

3. What could you do to be sure the important information on each page remains?

Definition/Information

1. TELOMERES: TTAGGG repeats

perhaps 2000 times!

2. In your body, shortening at the rate of about 31 bp/year

Definition/Information

1. 80 year old person, telomeres are about 5/8 length at birth

How can this be remedied?

1. Egg and SPERM cells and other cells in other organisms have overcome this problem

2. TELOMERASE Enzyme that catalyzes the addition of lost DNA by using an RNA template

How can this be remedied?

1. Stopwatch = Telomere deterioration

2. Germ cells never start the watch.

3. Natural selection has built our telomeres so they can survive at most 70-90 years.

4. People from long-lived families may have longer telomeres

How can this be remedied?

5. Remember the HeLa Cells?

6. Worldwide, weigh more than 400 times her own body weight

7. Oct. 11 is recognized as Henrietta Lacks Day in Atlanta

8. HeLa cells have excellent telomerase

Cancer Cells and Telomerase

• Switching on of telomerase genes is an essential mutation that must occur if a cancer is to turn malignant

Figure 16.18 The end-replication problem

Figure 16.19a Telomeres and telomerase: Telomeres of mouse chromosomes

Figure 16.19b Telomeres and telomerase