Mary K. Campbell
Shawn O. Farrell
international.cengage.com/
Chapter Ten
Biosynthesis of Nucleic Acids: Replication
Paul D. Adams • University of Arkansas
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Replication of DNA
• Naturally occurring DNA exists in single-stranded
and double-stranded forms, both of which can exist
in linear and circular forms
• Difficult to generalize about all cases of DNA
replication
• We will study the replication of circular doublestranded DNA and then of linear double-stranded
DNA
• most of the details we discuss were first investigated
in prokaryotes, particularly E. coli
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Flow of Genetic Information in the Cell
• Mechanisms by which information is transferred in
the cell is based on “Central Dogma”
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Prokaryotic Replication
• Challenges in duplication of circular double-stranded
DNA
• achievement of continuous unwinding and
separation of the two DNA strands
• arotection of unwound portions from attack by
nucleases that attack single-stranded DNA
• synthesis of the DNA template from one 5’ -> 3’
strand and one 3’ -> 5’ strand
• efficient protection from errors in replication
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Prokaryotic Replication (Cont’d)
• Replication involves separation of
the two original strands and
synthesis of two new daughter
strands using the original strands
as templates
• Semiconservative replication:
each daughter strand contains one
template strand and one newly
synthesized strand
• Incorporation of isotopic label as
sole nitrogen source (15NH4Cl)
• Observed that 15N-DNA has a
higher density than 14N-DNA,
and the two can be separated
by density-gradient
ultracentrifugation
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Evidence for Semiconservative Replication
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Which Direction does Replication go?
• DNA double helix unwinds at a specific point called an
origin of replication
• Polynucleotide chains are synthesized in both
directions from the origin of replication; DNA
replication is bidirectional in most organisms
• At each origin of replication, there are two replication
forks, points at which new polynucleotide chains are
forks
formed
• There is one origin of replication and two replication
forks in the circular DNA of prokaryotes
• In replication of a eukaryotic chromosome, there are
several origins of replication and two replication forks
at each origin
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Bidirectional Replication
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DNA Polymerase Reaction
• The 3’-OH group at the end of the growing DNA
chain acts as a nucleophile.
• The phosphorus adjacent to the sugar is attacked,
and then added to the growing chain.
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DNA Polymerase
•
DNA is synthesized from its 5’ -> 3’ end (from the 3’ -> 5’ direction of the template)
• the leading strand is synthesized continuously in the 5’ -> 3’ direction
toward the replication fork
• the lagging strand is synthesized semidiscontinuously (Okazaki
Okazaki
fragments) also in the 5’ -> 3’ direction, but away from the replication
fork
• lagging strand fragments are joined by the enzyme DNA ligase
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Properties of DNA Polymerases
• There are at least five types of DNA polymerase (Pol) in E
coli, three of which have been studied extensively
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Function of DNA Polymerase
• DNA polymerase function has the following
requirements:
• all four deoxyribonucleoside triphosphates: dTTP,
dATP, dGTP, and dCTP
• Mg2+
• an RNA primer - a short strand of RNA to which the
growing polynucleotide chain is covalently bonded in
the early stages of replication
• DNA-Pol I: repair and patching of DNA (remove and
fill up primers in lagging strand)
• DNA-Pol III: responsible for the polymerization of the
newly formed DNA strand
• DNA-Pol II, IV, and V: proofreading and repair
enzymes
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DNA Polymerase III
The "real" replicative polymerase in E. coli
It’s fast: up to 1,000 dNTPs added/sec/enzyme
It’s highly processive:
processive: >500,000 dNTPs added
before dissociating
It’s accurate: makes 1 error in 107 dNTPs
added, with proofreading, this gives a final
error rate of 1 in 1010 overall.
IT’S COMPLICATED!!!
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Supercoiling and Replication
• DNA gyrase (class II
topoisomerase) catalyzes
reaction involving relaxed
circular DNA:
• creates a nick in relaxed
circular DNA
• a slight unwinding at the
point of the nick
introduces supercoiling
• the nick is resealed
• The energy required for this
process is supplied by the
hydrolysis of ATP to ADP
and Pi
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Replication with Supercoiled DNA
• Replication of supercoiled circular DNA
• DNA gyrase has different role here. It introduces a
nick in supercoiled DNA
• a swivel point is created at the site of the nick
• the gyrase opens and reseals the swivel point in
advance of the replication fork
• the newly synthesized DNA automatically assumes
the supercoiled form because it does not have the
nick at the swivel point
• helicase
helicase, a helix-destabilizing protein, promotes
unwinding by binding at the replication fork
• single-stranded binding (SSB) protein stabilizes
single-stranded regions by binding tightly to them
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Primase Reaction
• The primase reaction
• RNA serves as a primer in DNA replication
• primer activity first observed in-vivo.
• Primase - catalyzes the copying of a short stretch of
the DNA template strand to produce RNA primer
sequence
• Synthesis and linking of new DNA strands
• begun by DNA polymerase III
• the newly formed DNA is linked to the 3’-OH of the
RNA primer
• as the replication fork moves away, the RNA primer is
removed by DNA polymerase I
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Replication Fork General Features
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Overview of DNA Replication
Synthesis of the lagging strand
1. Unwinding of parental duplex
by helicase with help of gyrase
and elongation of leading
strand by DNA polymerase III
expose single-strand region in
front of lagging strand
3. Polymerase III extends
DNA Okazaki fragment
from primer
4. Polymerase I eliminates
downstream RNA primer by
nick translation
2. Primase synthesizes
RNA primer
5. DNA ligase ligates
Okazaki fragment to rest of
lagging strand
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Proofreading and Repair
• DNA replication takes place only once each generation in
each cell
• Errors in replication (mutations) occur spontaneously only
once in every 109 to 1010 base pairs
• Can be lethal to organisms
• Proofreading - the removal of incorrect nucleotides
immediately after they are added to the growing DNA
during replication (Figure 10.10)
• Errors in hydrogen bonding lead to errors in a growing
DNA chain once in every 104 to 105 base pairs
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Proofreading Improves Replication Fidelity
• CutCut-and
and--patch catalyzed by Pol I: cutting is removal of the
RNA primer and patching is incorporation of the required
deoxynucleotides
• Nick translation:
translation Pol I removes RNA primer or DNA
mistakes as it moves along the DNA and then fills in
behind it with its polymerase activity
• Mismatch repair: enzymes recognize that two bases are
incorrectly paired, the area of mismatch is removed, and
the area replicated again
• Base excision repair: a damaged base is removed by
DNA glycosylase leaving an AP site; the sugar and
phosphate are removed along with several more bases,
and then Pol I fills the gap
• Nucleotide excition repair: damaged nucleotides that
lead to deformed DNA structures are removed as part of a
large section that contain the deformed structure
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DNA Polymerase Repair
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Mismatch Repair in Prokaryotes
• Mechanisms of mismatch repair encompass:
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Base excision repair
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Nucleotide excision repair
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DNA Recombination
• Genetic Recombination- When genetic information
is rearranged to form new associations
• Homologous - Reactions between homologous
sequences
• Nonhomologous- Different nucleotide sequences
recombine
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Recombination
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Eukaryotic Replication
• Not as understood as
prokaryotic. Due in no
small part to higher level
of complexity.
• Cell growth and division
divided into phases: M,
G1, S, and G2
• DNA replication takes
place during S phase
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Eukaryotic Replication
• Best understood
model for control of
eukaryotic replication
is from yeast.
• DNA replication
initiated by
chromosomes that
have reached the G1
phase
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Eukaryotic Replication
• ORC – origin recognition
complex
• RAP – replication activator
protein
• RLF – replication licensing
factors
• CDK – cyclin dependent
protein kinases
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Eukaryotic DNA Polymerase
• At least 15 different polymerases are present in
eukaryotes (5 have been studied more extensively)
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Structure of the PCNA Homotrimer
• PCNA (proliferating cell nuclear antigen) is the eukaryotic equivalent
of the part of Pol III that functions as a sliding clamp (β).
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The Eukaryotic Replication Fork
(single stranded binding protein)
(replication factor C)
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The Eukaryotic Replication Fork – initiation
of replication (in yeast)
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The Eukaryotic Replication Fork
• The general features of DNA replication in eukaryotes are
similar to those in prokaryotes. Differences summarized in
Table 10.5.
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Telomerase and Cancer (Biochemical Connections)
• Replication of linear DNA molecules poses particular
problems at the ends of the molecules
• Ends of eukaryotic chromosomes called telomeres
• Telomere- series of repeated DNA sequences
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