Download DNA: The Genetic Material

Chapter 14
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
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
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
Avery, MacLeod, & McCarty, 1944
repeated 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
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
- 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
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
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
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.
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
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
James Watson and Francis Crick, 1953
• deduced the structure of DNA using evidence from Chargaff, Franklin, and
others
• proposed a double helix 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
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.
Matthew Meselson & Franklin Stahl, 1958
investigated the process of DNA replication
considered 3 possible mechanisms:
• conservative model
• semiconservative model
• dispersive model
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
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
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.
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
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.
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
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
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.
• https://www.youtube.com/watch?v=27TxKoFU2Nw
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.
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.
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
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.
- DNA-damaging agents
- repair mechanisms
- specific vs. nonspecific mechanisms
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