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The Molecular Basis of Inheritance
CHAPTER 16: DNA STRUCTURE AND REPLICATION
You must know
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The structure of DNA
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The knowledge about DNA gained from the work of Griffith; Avery,
MacLeod, and McCarty; Hershey and Chase; Wilkins and Franklin;
and Watson and Crick
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Replication is semi-conservative and occurs 5’ to 3’
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The roles of DNA polymerase III, DNA polymerase I, ligase,
helicase, primase, and topoisomerase in replication
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The general differences between bacterial chromosomes and
eukaryotic chromosomes
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How DNA packaging can affect gene expression
DNA is the Genetic Material
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Evidence that DNA can transform Bacteria
Frederick Griffith was studying Streptoccoccus pneumonia, a
bacterium that causes pneumonia in mammals, trying to
develop a vaccine against pneumonia.
• Griffith had two strands (varieties), one pathogenic (diseasecausing) and one non-pathogenic (harmless)
• He killed the pathogenic bacteria using heat and mixed
them will the non-pathogenic bacteria and injected the
mixture into different mice.
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Frederick Griffith Experiment
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S-Strain= pathogenic
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R-Strain= nonpathogenic
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Transformation-a
change in genotype and
phenotype due to the
assimilation of external
DNA by a cell.
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What caused this
transformation?
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Bacteriologist, who spent 14 years
identifying the transforming
substance.
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Focused on 3 main candidates: DNA, RNA,
and proteins.
Avery broke open the heat-killed
pathogenic bacteria and extracted the
components.
Treated the samples with different agents
to kill one type of molecule, then tested
the samples ability to transform live
nonpathogenic bacteria.
Only when DNA was allowed to remain
active did the bacteria transform.
In 1944, Avery and his colleagues, Maclyn
McCarty and Colin MacLeod announced
their findings.
Oswald Avery
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Evidence that Viral DNA can Program Cells
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Bacteriphages (phage)- viruses that infect bacteria.
Virus- is little more than DNA enclosed by a protective coat
(protein).
To produce more viruses, a virus must infect a cell and take
over the cell’s metabolic machinery.
Alfred Hershey and Martha Chase performed experiments
showing that DNA is the genetic material of a phage known
as T2.
T2 infects E. coli, turning it into a T2 factory that releases
many copies when the cell reptured.
But which viral component-protein or DNA- was
responsible?
Hershey and Chase Experiment
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They used radioactive sulfur and
phosphorous to trace the fates of protein
and DNA.
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Results: When proteins were labeled (batch
1), radioactivity was found outside the cell.
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When DNA was labeled (batch 2),
radioactivity was found inside the cell.
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Bacterial cells with radioactive phage DNA
released new phages with some radioactive
phosphourus.
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Conclusion: DNA functions as the genetic
material of phage T2.
Building a Structural Model of DNA
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Each DNA nucleotide monomer consists of a
nitrogenous base (T, A, C or G), the sugar
deoxyribose, and a phosphate group.
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The phosphate group of one nucleotide is attached
to the sugar of the next, forming a “backbone” of
alternating phosphate and sugars from which the
bases project.
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The polynicleotide strand has directionality, from
the 5’ end (with the phosphate group) to the 3’ end
(with the –OH group of the sugar)
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5’ and 3’ refers to the numbers assigned to the
carbons in the sugar ring.
Watson and Crick
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Crick was studying the protein structure with a technique called X-ray crystallography.
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Watson saw an X-ray diffraction image of DNA produced by Rosalind Franklin, while
visiting Maurice Wilkins lab.
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Watson was familiar with this type of an X-ray diffraction pattern that helical molecules
produced, closer examination of the image confirmed that DNA was helical in shape.
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The photo implied that the helix was made of up two strands.
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Watson and Crick began making models of a double helix that conformed to the X-ray
measurements.
Franklin & Wilkins – early 1950s
Franklin deduced that DNA was helical and
consisted of 2 or 3 chains
James Watson and Francis Crick
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In 1953, they showed the world an
elegant double-helix model for the
structure of deoxyribonucleic acid.
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The (hydrophobic) nitrogenous bases in
the molecule’s interior.
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The negatively charged phosphate group
wouldn’t be forced into the interior.
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The two sugar-phosphate backbones are
antiparallel
DNA structure
• Sugar-phosphate
backbone with
rungs of
nitrogenous bases
• Strands are
antiparallel
1 nucleotide
Nitrogenous base pairing
Nitrogenous Bases:
-Purines – Adenine (A) and Guanine (G)
-Pyrimidines – Cytosine (C), Thymine (T), Uracil (U)
A Model for DNA replication: the basic concept
Semi-Conservative Replication
A. New strands are composed of 1 strand of parental
DNA and 1 strand of newly formed DNA
B. Free-floating nucleotides
1. Can be DNA or RNA
2. New complimentary DNA strands
are then synthesized by joining
together deoxyribonucleotide
triphosphates, one at a time, and
with the removal of a diphosphate.
Replication Enzymes
Enzyme
Helicase
Topoisomerase
SSBs
Primase
DNA Polymerase III
DNA Polymerase I
DNA Ligase
Function
Unzips & unwinds DNA
Relieves strain of unwound DNA
Help hold DNA open and stabilize it
Builds RNA Primer
Builds new DNA strand
Replaces RNA primer with DNA
Joins Okazaki fragments together
https://www.youtube.com/watch?v=OnuspQG0Jd0
1. Helicase unzips DNA (breaks hydrogen bonds) creating a
replication bubble at the origin of replication
Multiple origins per chromosome in eukaryotes
• Each side of bubble has replication fork
• Bubble enlarges as replication proceeds until bubbles meet
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2.
Primase builds a short primer (RNA chain) of RNA
nucleotides (5 – 10 bases)
3. DNA Polymerase III builds the complimentary strand of DNA in
the 5’  3’ direction
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Free-floating DNA nucleotides move in to match up with parent
strand, DNA polymerase III moves along and binds them together
4. DNA Polymerase I replaces RNA primer with DNA
nucleotides
Leading and Lagging Strands
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New nucleotides must be added on to the 3’ end
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Leading strand – Bases easily added as DNA is unzipped
Lagging Strand – has a delay
Section unzips, then strand is built back towards origin
• Results in chunks called Okazaki fragments
• DNA ligase bonds Okazaki fragments together after primer is replaced
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https://www.y
outube.com/
watch?v=Onu
spQG0Jd0
Speed and Accuracy
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~4000 nucleotides per second
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Mismatch repair – repair enzymes fix incorrectly placed
nucleotides
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Nucleotide excision repair – enzymes called nucleases cut
out incorrect nucleotides and then gap is filled in with
correct nucleotide
telomeres
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Lose a small portion of the chromosome every time it is replicated
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Telomeres consist of highly repetitive sequences in order to protect coding genes
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Cancer cells (ex. HeLa) – telomerase is activated – prevents degradation of telomeres and renders
cells “immortal”
Interesting article on HeLa cells:
http://berkeleysciencereview.com/
article/good-bad-hela/
DNA packaging
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Prokaryotes – one circular chromosome associated with very
few proteins
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Eukaryotes – linear chromosomes associated with many
proteins
• Histones – proteins that associate
with DNA to help it coil
• DNA is negatively charged, histones
are positively charged
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Chromatin – the packaged DNA and proteins
• The more tightly coiled the DNA is, the less accessible it is to
transcription enzymes = coils control gene expression
• Euchromatin – very extended and accessible to transcription
– form of most DNA during interphase
• Heterochromatin – more condensed (like during mitosis)
and generally not transcribed (Barr bodies are also an
example)