Chapter 16
Chapter
16 Basis of
Molecular
Inheritance
(DNA structure and
Replication)
Helicase Enzyme
The Amazing Race
What is the genetic material?
DNA or protein?
1928 Griffith – transformation of
pneumonia bacterium
1944 Avery –
further studied
transformation by
destroying lipids,
CHO, and
proteins
1947 Chargaff –
• Quantified purines
and pyrimidines
• Suggested base
pairing rules (A=T,
C=G)
1950 Wilkins and Franklin – DNA
X-rays
(a) Rosalind Franklin
(b) Franklin’s X-ray diffraction
photograph of DNA
1952 Hershey and Chase –
bacteriophages – incorporation of
radioactive viral DNA in new
phages
EXPERIMENT
Phage
Radioactive
protein
Bacterial cell
Batch 1:
radioactive
sulfur (35S)
DNA
Radioactive
DNA
Batch 2:
radioactive
phosphorus (32P)
EXPERIMENT
Phage
Empty
protein
Radioactive shell
protein
Bacterial cell
Batch 1:
radioactive
sulfur (35S)
DNA
Phage
DNA
Radioactive
DNA
Batch 2:
radioactive
phosphorus (32P)
EXPERIMENT
Phage
Empty
protein
Radioactive shell
protein
Radioactivity
(phage
protein)
in liquid
Bacterial cell
Batch 1:
radioactive
sulfur (35S)
DNA
Phage
DNA
Centrifuge
Pellet (bacterial
cells and contents)
Radioactive
DNA
Batch 2:
radioactive
phosphorus (32P)
Centrifuge
Pellet
Radioactivity
(phage DNA)
in pellet
1953 Watson and Crick – DNA
Model
1962 Nobel Prize awarded to
Watson and Crick and Wilkins
** Conclusion: DNA = Genetic
Material, not Protein
Models of DNA Replication
Semi-Conservative Model
(1950s - Meselson and Stahl)
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)
AntiParallel
Structure of
DNA
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 basepair 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.
5’
5’
3’
3’
5’
3’
3’
5’
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
Flash Overview
DNA i
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 overexposure 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
• Most multicellular organisms
do not have telomerases
that elongate telomeres
(humans don’t have them)
• So, telomeres = limiting
factor in life span of certain
tissues
• Older individuals typically
have shorter telomeres