DNA: The Genetic MaterialChapter 14

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The Genetic Material

Griffith’s results:

- live S strain cells killed the mice

- live R strain cells did not kill the mice

- heat-killed S strain cells did not kill the mice

- heat-killed S strain + live R strain cells killed the mice

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The Genetic Material

Frederick Griffith, 1928

studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia

there are 2 strains of Streptococcus:

- S strain is virulent

- R strain is nonvirulent

Griffith infected mice with these strains hoping to understand the difference between the strains

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The Genetic Material

Griffith’s conclusion:

- information specifying virulence passed from the dead S strain cells into the live R strain cells

- Griffith called the transfer of this information transformation

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The Genetic Material

Avery, MacLeod, & McCarty, 1944repeated Griffith’s experiment using purified

cell extracts and discovered:- removal of all protein from the transforming material did not destroy its ability to transform R strain cells- DNA-digesting enzymes destroyed all transforming ability- the transforming material is DNA

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The Genetic Material

Hershey & Chase, 1952

- investigated bacteriophages: viruses that infect bacteria

- the bacteriophage was composed of only DNA and protein

- they wanted to determine which of these molecules is the genetic material that is injected into the bacteria

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The Genetic Material

- Bacteriophage DNA was labeled with radioactive phosphorus (32P)

- Bacteriophage protein was labeled with radioactive sulfur (35S)

- radioactive molecules were tracked - only the bacteriophage DNA (as indicated

by the 32P) entered the bacteria and was used to produce more bacteriophage

- conclusion: DNA is the genetic material

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DNA Structure

DNA is a nucleic acid.

The building blocks of DNA are nucleotides, each composed of:

– a 5-carbon sugar called deoxyribose

– a phosphate group (PO4)

– a nitrogenous base

• adenine, thymine, cytosine, guanine

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DNA Structure

The nucleotide structure consists of

– the nitrogenous base attached to the 1’ carbon of deoxyribose

– the phosphate group attached to the 5’ carbon of deoxyribose

– a free hydroxyl group (-OH) at the 3’ carbon of deoxyribose

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DNA Structure

Nucleotides are connected to each other to form a long chain

phosphodiester bond: bond between adjacent nucleotides– formed between the phosphate group of

one nucleotide and the 3’ –OH of the next nucleotide

The chain of nucleotides has a 5’ to 3’ orientation.

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DNA Structure

Determining the 3-dimmensional structure of DNA involved the work of a few scientists:

– Erwin Chargaff determined that

• amount of adenine = amount of thymine

• amount of cytosine = amount of guanine

This is known as Chargaff’s Rules

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DNA Structure

Rosalind Franklin and Maurice Wilkins

– Franklin performed X-ray diffraction studies to identify the 3-D structure

– discovered that DNA is helical

– discovered that the molecule has a diameter of 2nm and makes a complete turn of the helix every 3.4 nm

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DNA Structure

James Watson and Francis Crick, 1953

– deduced the structure of DNA using evidence from Chargaff, Franklin, and others

– proposed a double helix structure

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DNA Structure

The double helix consists of:

– 2 sugar-phosphate backbones

– nitrogenous bases toward the interior of the molecule

– bases form hydrogen bonds with complementary bases on the opposite sugar-phosphate backbone

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DNA Structure

The two strands of nucleotides are antiparallel to each other

– one is oriented 5’ to 3’, the other 3’ to 5’

The two strands wrap around each other to create the helical shape of the molecule.

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DNA Replication

Matthew Meselson & Franklin Stahl, 1958

investigated the process of DNA replication

considered 3 possible mechanisms:

– conservative model

– semiconservative model

– dispersive model

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DNA Replication

Bacterial cells were grown in a heavy isotope of nitrogen, 15N

all the DNA incorporated 15N

cells were switched to media containing lighter 14N

DNA was extracted from the cells at various time intervals

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DNA Replication

The DNA from different time points was analyzed for ratio of 15N to 14N it contained

After 1 round of DNA replication, the DNA consisted of a 14N-15N hybrid molecule

After 2 rounds of replication, the DNA contained 2 types of molecules:

– half the DNA was 14N-15N hybrid

– half the DNA was composed of 14N

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DNA Replication

Meselson and Stahl concluded that the mechanism of DNA replication is the semiconservative model.

Each strand of DNA acts as a template for the synthesis of a new strand.

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DNA Replication

DNA replication includes:

– initiation – replication begins at an origin of replication

– elongation – new strands of DNA are synthesized by DNA polymerase

– termination – replication is terminated differently in prokaryotes and eukaryotes

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Prokaryotic DNA Replication

The chromosome of a prokaryote is a circular molecule of DNA.

Replication begins at one origin of replication and proceeds in both directions around the chromosome.

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Prokaryotic DNA Replication

The double helix is unwound by the enzyme helicase

DNA polymerase III (pol III) is the main polymerase responsible for the majority of DNA synthesis

DNA polymerase III adds nucleotides to the 3’ end of the daughter strand of DNA

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Unwinding

• As helicase unwinds the DNA, this causes supercoiling and stress.

• Topoisomerase enzymes and DNA gyrase help to relieve this stress and strain.

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Prokaryotic DNA Replication

DNA replication is semidiscontinuous.

– pol III can only add nucleotides to the 3’ end of the newly synthesized strand

– DNA strands are antiparallel to each other

leading strand is synthesized continuously (in the same direction as the replication fork)

lagging strand is synthesized discontinuously creating Okazaki fragments

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Okazaki fragments

• Fragments of DNA used to synthesize the lagging strand in direction moving away from the replication fork.

• These fragments are joined together to form the lagging strand by the enzyme ligase.

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Prokaryotic DNA Replication

The enzymes for DNA replication are contained within the replisome.

The replisome consists of

– the primosome - composed of primase and helicase

– 2 DNA polymerase III molecules

The replication fork moves in 1 direction, synthesizing both strands simultaneously.

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Eukaryotic DNA Replication

The larger size and complex packaging of eukaryotic chromosomes means they must be replicated from multiple origins of replication.

The enzymes of eukaryotic DNA replication are more complex than those of prokaryotic cells.

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Eukaryotic DNA Replication

Synthesizing the ends of the chromosomes is difficult because of the lack of a primer.

With each round of DNA replication, the linear eukaryotic chromosome becomes shorter.

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Eukaryotic DNA Replication

telomeres – repeated DNA sequence on the ends of eukaryotic chromosomes

– produced by telomerase

telomerase contains an RNA region that is used as a template so a DNA primer can be produced

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DNA Repair

- DNA-damaging agents

- repair mechanisms

- specific vs. nonspecific mechanisms

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DNA Repair

Mistakes during DNA replication can lead to changes in the DNA sequence and DNA damage.

DNA can also be damaged by chemical or physical agents called mutagens.

Repair mechanisms may be used to correct these problems.

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DNA Repair

DNA repair mechanisms can be:

– specific – targeting a particular type of DNA damage

• photorepair of thymine dimers

– non-specific – able to repair many different kinds of DNA damage

• excision repair to correct damaged or mismatched nitrogenous bases

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