chapter 16 molecular basis of inheritance (dna structure ... · molecular basis of inheritance (dna...
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Conclusions of Avery, et. al
• DNA is heritable agent.
• Results, unfortunately, were not enough to
sway the opinion of most scientists.
– How could one molecule cause so much
variation?
1950 Wilkins and Franklin – DNA
X-rays
(a) Rosalind Franklin (b) Franklin’s X-ray diffraction
photograph of DNA
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive
protein
Radioactive
DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive
protein
Radioactive
DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
Empty protein shell
Phage DNA
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive
protein
Radioactive
DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
Empty protein shell
Phage DNA
Centrifuge
Centrifuge
Pellet
Pellet (bacterial cells and contents)
Radioactivity (phage protein) in liquid
Radioactivity (phage DNA) in pellet
Significance of Hershey &
Chase Expt.• Unambiguously proved that DNA is the
agent of heredity, not proteins.
1962 Nobel Prize awarded to
Watson and Crick and Wilkins
** Conclusion: Now that we
know DNA’s structure, we can
figure out what it does and how it
does it.
Fun DNA Replication Facts
• 6 billion bases in human cell = 2 hours of
replication time
• 500 nucleotides added per second
• Accurate (errors only 1 in 10,000 base
pairs)
Mechanism of Replication
Step 1
• Origins of Replication = Special site(s) on DNA w/Specific sequence of nucleotides where replication begins
– Prokaryotic Cells = 1 site (circular DNA)
– Eukaryotic Cells = several sites (strands)
Steps 2 - 5• Helicase: (enzyme) unwinds
DNA helix forming a “Y”
shaped replication fork on
DNA
• Replication occurs in two
directions, forming a replication
bubble
• To keep strands separate,
DNA binding proteins attach
to each strand of DNA
• Topoisomerases: enzymes
that work w/helicase to prevent
“knots” during unwinding.
Step 6 - Priming
• Priming = due to physical limitation of DNA Polymerase, which can only add DNA nucleotides to an existing chain
• RNA primase – initiates DNA replication at Origin of Replication by adding short segments of RNA nucleotides.
• Later these RNA segments are replaced by DNA nucleotides by DNA Pol.
Step 7• DNA Pol. = enzyme that
elongates new DNA strand by adding proper nucleotides that base-pair with parental DNA template
• DNA Pol. can only add nucleotides to the 3’ end of new DNA, so replication occurs from a 5’ to 3’ direction
• Leading vs. Lagging Strand results
Leading vs. Lagging Strand
• Leading Strand: strand
that can elongate
continuously as the
replication for progresses
• Lagging Strand: strand
that cannot elongate
continuously and moves
away from replication fork.
• Short Okazaki fragments
are added from a 5’ to 3’
direction, as replication fork
progresses.
Step 8
• DNA Ligase = enzyme that “ligates” or covalently
bonds the Sugar-Phosphate backbone of the short
Okazaki fragments together
• Primers are required prior to EACH Okazaki
fragment
DNA i
Overview of DNA
Replication
Step 10: Fixing Errors
• DNA Pol. Proofreads as it elongates
• Special enzymes fix a mismatch nucleotide pairs
• Excision Repair:
– Nuclease: Enzyme that cuts damaged segment
– DNA Pol. Fills in gap with new nucleotide
Mutations
• Thymine Dimers (covalent bonding btwn
Thymine bases) –often caused by over-
exposure to UV rays DNA buckeling skin
cancer results, unless corrected by excision
repair
• Substitutions: incorrect pairing of nucleotides
• Insertions and Deletions: an extra or missing
nucleotide causes “frameshift” mutations
(when nucleotides are displaced one position)
Problems with Replication
• Since DNA
Polymerase can
only add to a 3’ end
of a growing chain,
the gap from the
initial 5’ end can not
be filled
• Therefore DNA gets
shorter and shorter
after each round of
replication
Solution?
• Bacteria have circular DNA (not a problem)
• Ends of some eukaryotic chromosomes have telomeres at the ends (repeating nucleotide sequence that do not code for any genes)
• Telomeres can get shorter w/o compromising genes
• Telomerase = enzyme that elongates telomeres since telomeres will shorten
Telomerases are not in most
organisms
• Many multicellular
organisms do not have
telomerases that elongate
telomeres (humans have
them in some cells)
• So, telomeres = limiting
factor in life span of certain
tissues
• Older individuals typically
have shorter telomeres