Chapter 16 The Molecular Basis of Inheritance

Outline

• DNA is the Genetic Material

• The Structure of DNA

• DNA Replication and Repair

The Requirements of Genetic Material

• It must be able to replicate, in order to be in each cell of a growing organism.

• It must be able to control expression of traits.

• The genetic material must be able to encode the sequence of proteins.

• It must be able to change over time.

• Mutations are the raw material for evolution.

What is the Genetic Material?

• Genes are located on chromosomes.

• Chromosomes are composed of both DNA and proteins.

What is the Genetic Material?

• It was proposed that proteins carried the genetic information and that the DNA served as a scaffold.

– Proteins are composed of 20 different subunits so are very diverse and have great specificity of function.

What is the Genetic Material?

• A series of experiments lead to the deduction that DNA, not proteins are the genetic material.

The Griffith Experiment (Fig 16.2)

• A mixture of heat-killed (coated) pathogenic and live (coatless) nonpathogenic strains of S. pneumoniae killed mice.

• The blood of the mice contained live pathogenic (coated) strain of S. pneumoniae with cell surface proteins from the nonpathogenic strains.

• Hereditary information was passed from the dead pathogenic bacteria, to the live, nonpathogenic bacteria, transforming them into pathogenic bacteria.

The Griffith Experiment

What is the Transforming Principle?

• The term transformation refers to a change in the genotype and phenotype of a cell due to uptake and assimilation of external DNA.

• Griffiths findings led Oswald Avery on a 14 year search to discover the identity of the transforming substance in Griffith’s experiments.

Avery, MacLeod, and McCarty

• 99.98% of proteins were removed from the transforming principle, but it still had transforming activity.

• The purified “transforming principle” was analyzed chemically and found to be DNA.

• Removing lipid, protein, and RNA did not affect the “transforming principle’s” activity.

• DNAses destroyed all transforming activity.

The Hershey-Chase Experiment

• T2 bacteriophage are viruses that infect bacterial cells.

The Hershey-Chase Experiment

• One set of viruses had viral proteins labeled with ³⁵S.

• Another set had viral DNA labeled with ³²P.

• ³⁵S radioactivity did not enter infected bacterial cells and ³²P radioactivity did.

• Viral DNA, not protein was responsible for directing the production of new viruses.

Figure 16.4 The Hershey-Chase experiment

Chargaff’s Rules

• It was know that DNA was a polymer of nucleotides with four bases, adenine (A), thymine (T), guanine (G), and cytosine (C).

• Erwin Chargaff analyzed the base content of the DNA from a number of species.

• He found that in the DNA of any one species the amounts of the four bases are not equal, but there is a characteristic ratio:

– The amount of adenine is the same as the amount of thymine.

– The amount of guanine is equal to the amount of cytosine.

What is the Genetic Material?

• Griffith, 1928: Hereditary information can pass between organisms.

• Avery, McCarty, & MacLeod, 1944: DNA is the “transforming principle”

• Hershey-Chase, 1952: nucleic acid, not protein, infects bacterial cells.

• Chargaff’s rules, 1947’s: A=T; G=C

Components of DNA

• Composed of units called nucleotides containing a sugar attached to a phosphate and a nitrogen containing base.

• Linked between the 5’ phosphate and the 3’ hydroxyl groups (phophodiester bond), to form a polymer.

• The proportion of A = T, and the proportion of G = C (Chargaff’s rules).

Figure 16.5 The structure of a DNA stand

Figure 16.8 Base pairing in DNA

Figure 16.7 The double helix

3D-Structure of DNA

• Chargoff’s Rules (A=T, G=C)

• Franklin’s X-ray diffraction Patterns of DNA fibers

– DNA is helical

– 2 nm diameter

– Complete helical turn every 3.4 nm

• Watson and Crick

– Model of the Double Helix

Unnumbered Figure (page 298) Purine and pyridimine

DNA Replication

"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."

Figure 16.9 A model for DNA replication: the basic concept

Semiconservative Replication

• Parent molecule serves as a template for replication.

• Each strand pairs with new nucleotides according to the rules of complimentary base pairing.

• The daughter molecules are hybrids, having one strand from the parent molecule, and another composed of new nucleotides

How do we know that DNA is replicated semiconservatively?

• The Meselson-Stahl Experiment

Figure 16.10 Three alternative models of DNA replication

The Meselson-Stahl Experiment: Protocol

• Bacterial cells grown for several generation in a medium w/ ¹⁵N.

• Transferred to medium w/ ¹⁴N.

• Samples of the bacteria were collected at several time points.

• DNA isolated from bacterial and separated according to weight by centrifuging in CsCl.

Figure 16.11 The Meselson-Stahl experiment tested three models of DNA replication

The Meselson-Stahl Experiment: Results

• Parent DNA had one heavy band, indicating that both strands are heavy.

• F₁ generation had one intermediate band, indicating that both strands were a hybrid between heavy and light.

• F₂ generation had one intermediate and one light band, indicating one light (unlabeled) molecule and another heavy/light hybrid.

• Indicated semiconservative replication.

The Replication Process

• Replication begins at special sites called replication origins.

• The genome of a bacterial cell has one replication origin.

– The replication origin has a particular DNA sequence that is recognized by a set of proteins that initiate replication.

– The two strands are pulled apart to form a ‘bubble’.

– Replication proceeds in both directions until the entire molecule is copied.

Origins of Replication

• Eukaryotic chromosomes have multiple origins.

– The replication bubble eventually fuse, rapidly copying the very long DNA molecules.

Figure 16.12 Origins of replication in eukaryotes

The Replication Process
(in E. coli)

• DNA polymerases catalyze the addition of nucleotides to the growing chain.

• Replication only begins at replication origins.

• New nucleotides base-pair with the nucleotides on the template.

• A phosphodiester bond is formed between the first phosphate of the incoming nucleotide, and the 3’ end of the previous nucleotide.

• Replication always progresses 5’ to 3’.

Figure 16.13 Incorporation of a nucleotide into a DNA strand

The strand always grows 5’ to 3’.

• The replication process is fueled by cleaving the high energy phosphate bond between the first and second phosphates of the incoming nucleotide.

• The substrate recognized by the DNA polymerase must include the 3’OH group.

Figure 16.14 The two strands of DNA are antiparallel

Okazaki Fragments

• A growing chain always elongates 5’ to 3’.

• Leading strand elongates toward the replication fork.

• Lagging strand elongates away from the replication fork.

– Synthesized discontinuously as a series of short segments that are later connected by DNA ligase.

– These segments are called Okazaki fragments.

Figure 16.15 Synthesis of the lagging strand

DNA Polymerase needs an RNA primer.

• DNA polymerases cannot initiate synthesis of a polymer – they can only elongate an existing strand.

• DNA polymerases recognize the 3’ hydroxyl group of an RNA primer.

• The primer is a short segment of RNA which is laid down by a primase.

Table 16.1 The main proteins of DNA replication and their functions

Steps of the Replication Process

1. Opening up the DNA double helix

- Initiator protein bind to replication origin

- Helicases unwind the duplex

- Single stand binding proteins stabilize the open strands

2. A primase builds an RNA primer

Figure 16.16 A summary of DNA replication

Steps of the Replication Process, con’t

3. DNA polymerase III assembles complementary strands.

- Leading and lagging strands

4. The primer is removed by DNA polymerase I, which fills in the gap, as well as gaps between Okazaki fragments.

5. DNA ligase joins the Okazaki fragments.

Figure 16.16 A summary of DNA replication

DNA polymerases proofread DNA during its replication and repair damage in existing DNA

• DNA polymerase has proofreading and editing activity

– 3’-5’ exonuclease.

• There are mechanisms to repair mistakes in replications or mutations that occur due to DNA damage.

– Mismatch repair

– Base excision repair

– Nucleotide excision repair

Figure 16.17 Nucleotide excision repair of DNA damage

The problem at the Ends

Figure 16.18 Shortening of the ends of linear DNA molecule.

Figure (not in text) Telomeres and telomerase

Figure 16.19 Telomeres and telomerase: Telomeres of mouse chromosomes

The End.