Transcript DNA Replication

DNA Replication
A.
B.
C.
DNA replication is semiconservative
DNA replication in E. coli
DNA replication in eukaryotes
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A. . . . Semiconservative

In DNA replication, the two strands of a
helix separate and serve as templates
for the synthesis of new strands
(nascent strands), so that one helix
gives rise to two identical “daughter”
helices
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A. . . . Semiconservative

Hypothetically, there could be three possible
ways that DNA replication occur:
– Conservative replication: One daughter helix gets
both of the old (template) strands, and the other
daughter helix gets both of the new (nascent)
strands
– Semiconservative: Each daughter helix gets one
old strand and one new strand
– Dispersive: The daughter helices are mixes of old
and new
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A. . . . Semiconservative

Two major lines of experiment in the mid
1950s – early 1960s demonstrated that DNA
replication is semiconservative, both in
prokaryotes and eukaryotes:
– Meselson and Stahl demonstrated
semiconservative replication in Escherichia coli in
1958
– Taylor, Woods, and Hughes demonstrated
semiconservative replication in Vicia faba (broad
bean) in 1957
– Experiments with other organisms support
semiconservative replication as the universal
mode for DNA replication
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B. Replication in E. coli
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DNA replication is semiconservative
and requires a template
Deoxynucleoside triphosphates
(dNTPs) (dATP, dTTP, dGTP, dCTP)
are the “raw materials” for the addition
of nucleotides to the nascent strand
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B. Replication in E. coli
Nucleotides are added only to the 3´
end of a growing nascent chain;
therefore, the nascent chain grows only
from the 5´ 3´ direction
 The addition of nucleotides to a growing
chain is called chain elongation
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B. Replication in E. coli
Addition of nucleotides to a nascent
chain is catalyzed by a class of
enzymes called DNA-directed DNA
polymerases (or DNA polymerases, for
short)
 E. coli has three DNA polymerases (I, II,
and III)
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B. Replication in E. coli
– DNA polymerase I was discovered in the
mid 1950s by Arthur Kornberg (it was
originally simply called “DNA polymerase”
– DNA polymerase I has three different
enzymatic activities:
5´ 3´ polymerase activity (elongation)
3´ exonuclease activity (proofreading function)
5´ exonuclease activity (primer excision)
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B. Replication in E. coli
– The 3´ exonuclease activity of DNA
polymerase I performs a “proofreading”
function: it excises mismatched bases at
the 3´ end, reducing the frequency of
errors (mutations)
– The 5´ exonuclease activity is responsible
for RNA primer excision (see later . . .)
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B. Replication in E. coli
– By the late 1960s, biologists suspected
that there must be additional DNA
polymerases in E. coli (to account for the
rate of replication observed in experiments)
– In the early 1970s, DNA polymerases II
and III were discovered
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B. Replication in E. coli
– DNA polymerases II and III each have two
enzymatic activities:
5´ 3´ polymerase activity (elongation)
3´ exonuclease activity (proofreading)
– Neither has the 5´ exonuclease activity
– DNA polymerase III is the enzyme
responsible for most of the nascent strand
elongation in E. coli
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B. Replication in E. coli

DNA polymerase can only elongate existing
chains; it cannot initiate de novo chain
synthesis
– Nascent strand initiation requires the formation of
a short RNA primer molecule
– The RNA primers are synthesized by RNA
primase (a type of 5´ 3´ RNA polymerase,
capable of initiating nascent chain synthesis from
a DNA template; uses ribose NTPs as nucleotide
source)
– The primers are eventually excised by the 5´
exonuclease activity of DNA polymerase I
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B. Replication in E. coli
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Replication begins at a location on the
chromosome called the origin of replication
(ori), and proceeds bidirectionally.
 As the DNA helix unwinds from the origin, the
two old strands become two distinctive
templates:
– the 3´ 5´ template,
– and the 5´ 3´ template
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B. Replication in E. coli
– Replication on the 3´ 5´ template is continuous
(leading strand synthesis), proceeding into the
replication fork
– Replication on the 5´ 3´ template is
discontinuous, resulting in the synthesis of short
nascent segments (lagging strand or Okazaki
fragments), each with its own primer
– After primer excision is complete, nascent
segments are “sealed” (the final phosphodiester
bond is formed) by DNA ligase
– DNA polymerase III may be able to synthesize
both the leading and lagging strands
simultaneously by having the 5´ 3´ template to
fold back.
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B. Replication in E. coli
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Several proteins are required to unwind
the helix
– Helicases
• dnaA protein recognizes the origin , binds, and
begins the separation of the helix
• dnaB dissociates from dnaC; the dnaB is
responsible for moving along the helix at the
replication fork, “unzipping” the helix
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B. Replication in E. coli
– DNA gyrase
• Makes temporary single-stranded “nicks”
(single PDE bond breaks) in one of the two
template strands to relieve the torsional stress
and supercoiling caused by the unwinding of
the helix
– Single-stranded binding proteins (SSBPs)
• Bind to the unwound strands of the template,
stabilizing the single-stranded state long
enough for
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http://www.mcb.harvard.edu/Losick/ima
ges/TromboneFinald.swf
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C. Eukaryotic DNA Replication
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Eukaryotic chromosomes have multiple
origins of replication on each chromosome
 There are 6 different eukaryotic DNA
polymerases
a,d,and eare essential for replication
band zare involved in repair
g is only active in mitochondrial DNA replication
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C. Eukaryotic DNA Replication
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Eukaryotic chromosomes are linear, not
circular like prokaryotic chromosomes
– The ends of eukaryotic chromosomes are
formed by an enzyme called telomerase
– Telomerase adds repeats of TTGGGG to
the 3´ ends of eukaryotic chromosomes
– The repeats fold over into a “hairpin”
structure, providing a primer for completion
of the end (telomere) structures
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C. Eukaryotic DNA Replication
– In most eukaryotic somatic cells, the
telomerase activity stops shortly after the
cell differentiates.
– After this, the chromsomes gradually
shorten with each division
– The loss of telomerase activity is a major
factor in cell aging
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How do genes work?
Genes carry the
instructions for making
and maintaining an
individual
 But how is this
information translated into
action?
 How does an organism’s
genotype specify its
phenotype?
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RR=
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Garrod
Provided the first clue to gene function
 studied alkaptonuria, a disease in
which homogentisic acid is secreted in
the urine.
 Hypothesized that the metabolic
pathway in which homogentisic acid is
an intermediate must be blocked in
alkaptonurics
 Block due to lack of an enzyme that
breaks down homogentisic acid,
leading to its buildup.
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Garrod
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George Beadle and Edward Tatum
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Developed the one-gene, one-enzyme
hypothesis from Garrod’s work
– Each gene carries the information for one
protein or enzyme.
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Did experiments using red bread mold
Neurospora crassa
– Irradiated mold to create mutants
– Tested if they could grow on minimal
media
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Adrian Srb and Norman Horowitz tested
for arginine
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Beadle and Tatum proposed: “One Gene-One Enzyme
Hypothesis”
However, it quickly became apparent that…
1. More than one gene can control each step in a pathway
(enzymes can be composed of two or more polypeptide
chains, each coded by a separate gene).
2. Many biochemical pathways are branched.
“One Gene-One Enzyme Hypothesis”
“One Gene-One Polypeptide Hypothesis”
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