The Molecular Basis of Inheritance

By the 1940’, scientists knew that chromosomes carried hereditary material and consisted of DNA & protein.

There were two experiments that proved that it was DNA only.

Frederick Griffith (1928)

He was trying to find a vaccine against Sterptococcus pneumonidae (bacteria that causes pneumonia in mammals).

Used 2 strains of bacteria ‘S’ and ‘R’.

Showed that bacteria could undergo transformation: bacteria taking up naked DNA for the surrounding environment.

Figure 16.1 Transformation of bacteria

Avery (1944)

Performed Griffith’s experiments and took it even farther.

Concluded DNA was the hereditary material, but was still met with skepticism.

Hershey & Chase (1952)

Discovered that DNA was the genetic material of a phage (virus) known as T2.

Hershey and Chase

Searching for Genetic Material

Hershey and Chase

√ bacteriophages (phages)

√ Expt: sulfur(S) is in protein, phosphorus (P) is in DNA; Used radioactive S to label protein, and radioactive P to label DNA

only P was found in host cell

Concluded that DNA, not protein, is the hereditary material

Figure 16.2ax Phages

Figure 16.2a The Hershey-Chase experiment: phages

Chargaff

DNA varies from species to species

Bases present in characteristic ratio

ratio of nucleotide bases (A=T; C=G)

“Chargaff’s Rules”

Watson & Crick (1953)

Discovered that DNA was a double helix.

The helix was ladder-like with a sugar-phosphate backbone.

The 2 backbones were antiparallel; they ran in opposite directions.

Figure 16.0 Watson and Crick

Summary

Mendel and Morgan – inheritance

Griffith – transformation

Avery et al. – DNA was transforming agent

Hershey and Chase – phage experiment proved DNA was agent

Chargaff – ratio of A = T, C = G

Pauling – three stranded helix

Wilkins and Franklin – X-ray crystallography

Watson and Crick

X-ray crystallography – helix shape, width of helix, spacing of nitrogen bases

2 strands

Sugar-phosphate sides, base rungs

Spacing was right for purine – pyrimidine pairing (Chargaff’s rules)

1 turn every .34nm (10 base pairs)

Structure lends itself to replication

Figure 16.5 The double helix

Figure 16.12 The two strands of DNA are antiparallel

There are 4 nitrogenous bases:

Adenine (A) purine

Guanine (G) purine

Thymine (T) pyrimidine

Cytosine (C) pyrimidine

Purines bond with pyrimidines.

A = T (2 bonds) and C = G (3 bonds)

Figure 16.6 Base pairing in DNA

Figure 16.3 The structure of a DNA stand

Since A always bond with T, their amounts in a strand of DNA are equal (same for G-C).

Weak hydrogen bonds hold the two strands of DNA together.

DNA replication

There are two strands that both need to be replicated.

Before replication, these strands must be separated.

Once separated, these strands act as the template for assembling a complementary strand.

3 Hypotheses for Replication:

1. Conservative

2. Semiconservative

3. Dispersive

1. Conservative

The parental double helix should remain intact and the 2 nd (new ) double helix is made from entirely new material.

2. Semiconservative

Each of the 2 resulting DNA molecules are composed of one original template and one newly created strand.

3. Dispersive

Both strands of each new helix contain both a mixture of old and new DNA.

Figure 16.8 Three alternative models of DNA replication

DNA replication is done in the semiconservative fashion; the other 2 hypotheses are not correct.

Meselson-Stahl Experiment

The process of DNA replication is:

Complex: The helix untwists as it copies its two antiparallel strands simultaneously. This requires the cooperation of over a dozen enzymes & proteins.

The process of DNA replication is:

Extremely Rapid: In prokaryotes, up to 500 nucleotides are added per second. It takes only a few hours to copy the 6 billion bases of a single human cell.

The process of DNA replication is:

Accurate: Only about one in a billion nucleotides is incorrectly paired.

Replication must start at specific sites. They are called the origins of replication.

These origins have a specific nucleotide sequence.

Specific proteins must bind to the origins to initiate replication.

In addition to proteins at the origin, a primer is needed to “prime” the rxn.

Primer = a short RNA segment that is complementary to a DNA segment.

Primers are short segments of RNA polymerized by an enzyme called primase.

The DNA helix opens up at an origin and a replication fork is created.

DNA helicase is responsible for opening up the fork.

The forks spread in both directions away from the central initiation point creating a replication bubble.

Vocabulary review

Origin of replication (“bubbles”): beginning of replication

Replication fork: ‘Y’-shaped region where new strands of DNA are elongating

Helicase: catalyzes the untwisting of the DNA at the replication fork

DNA polymerase: catalyzes the elongation of new DNA

Prokaryotic cells (and viral DNA) only have one origin.

Eukaryotic DNA has many origins creating many forks = many bubbles.

Enzymes called DNA polymerases (DNA pol) catalyze the synthesis of a new strand.

DNA pol links the nucleotides to the growing strand.

ALL DNA must be replicated in the 5’ to 3’ direction. (5’ à 3’)

Leading Strand

Continuous synthesis of both DNA strands at a replication fork in the same direction is not possible because DNA pol replicates 5’ à 3’

The problem is solved by the continuous synthesis of one strand, the leading strand, and discontinuous synthesis of the lagging strand.

Leading strand can continue in 1 direction.

The lagging strand

It is produced as a short series of segments called Okazaki fragments which are individually made in the 5’ à 3’ direction.

O. fragments are 1,000 to 2,000 nucleotides long in prokaryotes and 100 to 200 long in eukaryotes.

The lagging strand has many RNA primers.

Lagging strand

When the lagging strand is complete, RNA primers are removed by DNA pol and replaced with DNA.

Then they are linked together by an enzyme called DNA ligase.

Enzymes proofread DNA during its replication and repair damage in existing DNA.

Mismatch repair

Excision repair

Mismatch Repair

Corrects mistakes when DNA is synthesized.

DNA pol and other proteins assist in this process.

A heredity defect in one of these proteins has been found with one form of colon cancer.

In the absence of proofreading, errors accumulate.

Excision Repair

Corrects accidental changes that occur in existing DNA.

Changes can result from UV light, cigarette smoke, etc.

There are more than 50 types of DNA enzymes that repair damage.

DNA Repair

Mismatch repair: DNA polymerase

Excision repair: Nuclease

Telomere ends: telomerase