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CUBT105: MOLECULAR BIOLOGY
DNA Replication (Synthesis) &
Repair
TENDAI WALTER SANYIKA
CUT, DEPARTMENT OF BIOTECHNOLOGY
BLOCK 11, ROOM 10
Objectives
1. To describe the mechanism of DNA replication.
2. To describe the mechanisms of DNA repair.
3. To describe the structure and functions of E. coli DNA polymerases.
4. To explain the functions of the other enzymes involved in DNA replication and repair.
5. To explain the properties of DNA replication.
Genome Maintenance • Dedicated to the DNA structure and the
processes that propagate, maintain and alter
the genome from one cell generation to the next.
• Mostly involves DNA replication and repair.
• All DNA is replicated from a particular origin
known as a replicon.
• Two components control the initiation of
replication.
– The Replicator (sequences on DNA) and
– The Initiator (proteins that bind DNA to initiate the process).
DNA Replication
DNA Replication
• Replicator: – Entire site of cis-acting
DNA sequences
sufficient to direct the
initiation of DNA
replication.
• Initiator protein: – Specifically recognizes
a DNA element in the
replicator and activates
the initiation of
replication.
DNA Replication The Initiator Protein
• Three different functions of initiator protein:
– Binds to replicator,
– Distorts/unwinds a region of DNA,
– Interacts with and recruits additional replication
factors.
• In prokaryotes.
– DnaA in E. coli, which does all the 3 functions.
• In eukaryotes.
– Mainly the Origin Recognition Complex (ORC).
DNA Replication Watson & Crick, 1953
• "It has not escaped our notice that the specific
(base) pairing we have postulated immediately
suggests a possible copying mechanism for the
genetic material."
• The mechanism:
– Strand separation, followed by copying of
each strand.
• Each separated strand acts as a template for
the synthesis of a new complementary strand.
– DNA replication is semi-conservative.
DNA Replication DNA Polymerase
• In 1957, Arthur Kornberg and colleagues
demonstrated the existence of a DNA
polymerase, DNA polymerase I.
• DNA polymerases. – Enzymes required for DNA synthesis (or replication)
and/ or repair.
• DNA Pol I needs the following to replicate DNA: – All four deoxynucleotides (dNTPs).
– A template and
– A primer. – A single stranded DNA (ssDNA) primer with a free 3'-
OH that pairs with the template to form a short double
stranded region.
DNA Polymerases
DNA Polymerases Structure & Function: Prokaryotic vs Eukaryotic
DNA Polymerase I Replication Occurs In 5′3′ Direction
• Replication occurs 5' to 3'. – Nucleotides are added at the 3'-end of the growing
strand, beginning from the primer.
– Thus, DNA pol I has the 5' 3' polymerase activity.
• DNA Pol I catalyzes about 20 cycles of
polymerization before the new strand
dissociates from template. – 20 cycles constitutes moderate "processivity“.
– Pol I from E. coli is 928 aa (109 kD) monomer.
• In addition to 5'-3' polymerase activity, it has: – The 3'-5' exonuclease activity and
– The 5'-3' exonuclease activity.
• DNA Pol I resembles a hand that grips the
primer-template junction.
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Enzyme is a single-chain protein that
requires magnesium as a cofactor.
Each of its three enzymatic
activities are located into distinct
domains of the holoenzyme.
DNA Polymerase I The Palm Domain
• Contains two catalytic sites. – Polymerization site for addition of dNTPs.
– Exonuclease proofreading site for removal of the
mispaired dNTPs.
• The polymerization site: – Binds to two metal ions that alter the chemical
environment around the catalytic site and lead to the
catalysis.
– Monitors the accuracy of base-pairing for the most
recently added nucleotides by forming extensive
hydrogen bond contacts with minor groove of the newly
synthesized DNA.
• Exonuclease site/proof reading site.
DNA Polymerase I Mechanism of The Palm Domain
DNA Polymerase I The Finger Domain
• Binds to the incoming dNTP.
• Encloses the correct paired dNTP to the
position for catalysis.
• Bends the template to expose the only
nucleotide ready for forming base pairing with
the incoming nucleotide.
• Stabilizes the pyrophosphate during formation
of the phosphodiester bond.
DNA Polymerase I The Thumb Domain
• Not directly involved in catalysis but structural.
• Interacts with the synthesized DNA to maintain
correct position of the primer and the active
site.
• Also maintains a strong association between
DNA Pol and its substrate.
DNA Polymerase I Other Characteristics
• The processivity (rate) of DNA Pol. – The average number of nucleotides added each
time the enzyme binds a primer-template junction. – Varies from a few to >50,000 nucleotides.
• Rate of DNA synthesis related to processivity. – Rate-limiting step is the initial binding of polymerase
to the primer-template junction.
• Wrong dNTPs occasionally misincorporaed
due to high processivity. – Mismatched dNTPs removed by proofreading
exonuclease activity. – May be an integral activity of the DNA pol I.
– Relies on kinetic selectivity mechanism.
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• The 3' 5' exonuclease activity. – Serves a template dependent proofreading function.
– Removes nucleotides operating from the 3' end.
– Removes incorrectly matched bases. – The polymerase can pair residues again.
• The 5' 3' exonuclease activity. – Proofreads operating from the 5' end of DNA.
– Works together with the polymerase in the same
direction.
–Also involved in "nick translation“.
• Without proofreading error rate (mutation rate)
is 1 x 10-6. – With proofreading error rate decreases a1000-fold.
DNA Polymerase I Exonuclease Activity
DNA Polymerases Proofreading Mechanisms
• Uses a single site to catalyze the addition of
any of the four dNTPs.
• Recognizes the different dNTPs by monitoring
the ability of incoming dNTP to form A-T and
G-C base pairs.
– Incorrect base pair dramatically lowers the rate of
catalysis (kinetic selectivity).
– Hence will not be preferentially incorporated.
Distinguishing different dNTPs: kinetic selectivity
DNA Polymerases Proofreading Mechanisms
• Distinguishing between rNTP and dNTP by
steric exclusion of rNTPs from the active site.
• Enzymatic cleavage of DNA Pol I: – Subtilisin or trypsin cleavage.
– Produces two fragments. – A small fragment with 5‘ 3' exonuclease activity.
– A large fragment with both: » The 5' 3' polymerase and
» The 3' 5' exonuclease activities.
– Large subunit known as the "Klenow fragment”
(named after the discoverer, Hans Klenow).
• Klenow is the larger fragment generated from
the enzymatic cleavage of DNA polymerase I
of E. coli. – Very important for recombinant DNA applications.
– Properties to be discussed under that topic.
DNA Polymerase I The Klenow Fragment
DNA Polymerase I Klenow Fragment: Structure & Function
• T4 is a bacteriophage that infects E. coli. • Produces an enzyme T4 DNA polymerase.
• Functions of T4 DNA polynerase similar to Klenow
but:
– The 3′ → 5′ exonuclease activity of T4 DNA
polymerase is roughly 200 times higher.
– Klenow fragment will displace downstream
oligonucleotides as it polymerizes, T4 DNA
polymerase cannot.
DNA Polymerase I Klenow Similar to T4 DNA Polymerase
DNA Polymerase I Sliding Clamps
• Proteins that encircle the newly synthesized
double-stranded DNA and the polymerase. – Associated with the primer-template junction.
• Dramatically increase DNA polymerase activity. – Ensures the rapid rebinding of DNA Pol to the
same primer-template junction. – Increases the processivity of Polymerases.
• Eukaryotic sliding DNA clamp is PCNA.
– Proliferating cell nuclear antigen (PCNA).
DNA Polymerase I Sliding Clamps
• Clamp loader
– A class of protein complex.
– Catalyze the opening and placement of sliding
clamps.
– Operates on the DNA at primer-template junctions.
• Removal of sliding clamps.
– Only removed from the DNA once all the associated
enzymes complete their function.
DNA Polymerase I Sliding Clamps & Clamp Loaders
DNA Polymerases Specialized Roles in Different Cells
• Each organism has a distinct set of different
DNA Polymerases.
• Different organisms have different DNA
Polymerases.
• Prokaryotic Polymerases (E. coli)
– DNA Pol III holoenzyme: – A protein complex responsible for E. coli genome
replication.
– DNA Pol I: – Single subunit protein that removes RNA primer.
• Eukaryotic cells have multiple polymerases. – Three are essential to duplicate the genome:
– DNA Pol d, DNA Pol e and DNA Pol a (primase).
DNA Polymerase II (Pol II)
• A 90KD polypeptide enzyme.
– Single subunit protein.
• Mainly involved in 5′ 3′ repair synthesis.
– Polymerase activity in that direction.
• Has 3′ 5′ exonuclease activity which shows
its involvement in repair.
• It has no 5′ 3′ exonuclease activity.
• Can seal Okazaki fragments and carry out the
functions of Pol I in its absence.
DNA Polymerase III (Pol III) Main Replicating Polymerase in E. coli
• Has at least 10 different subunits.
• "Core" enzyme has three subunits - a, e, and – Alpha subunit.
– Polymerase activity.
– Epsilon subunit. – 3'-exonuclease activity.
– Theta subunit. – Function is unknown
– The beta subunit. – A dimer that forms a ring around DNA.
• Enzyme has enormously high processivity. – 5 million bases!
Prokaryotic DNA Polymerases In E. coli
Features of Replication Mostly in E. coli, but Many Features Are General
• Replication is bidirectional.
• The double helix must 1st be unwound.
– By helicases
• Supercoiling must be compensated.
– By DNA gyrases.
DNA Replication Features
• DNA polymerase I catalyzes formation of
phosphodiester bond. – Between 3’-OH of the deoxyribose (on the last nucleotide) &,
– the 5’-phosphate of the dNTP.
• Energy for DNA replication derived from the
release of two of the three phosphates (ppi) of
the dNTP.
• DNA polymerase “finds” the correct
complementary dNTP at each step in the
lengthening process. ‒ Rate ≤ 800 dNTPs/second.
‒ Low error rate.
• Direction of synthesis is 5′ to 3′.
Features of DNA Replication Semi-conservative
DNA Replication
Features of DNA Replication The Semi-conservative Model
• Matthew Meselson and Franklin Stahl.
– Showed that DNA replication results in new
DNA duplex molecules in which one strand is
from the parent duplex and the other is
completely new.
• Study involved isotope labelling and density
gradient centrifugation experiments.
• Meselson & Stahl Experiment (1958).
– Equilibrium density gradient centrifugation
differentially sediments DNA according to molecular
weight.
Features of DNA Replication The Semi-conservative Model
Features of DNA Replication The Semi-conservative Model
Features of DNA Replication Bidirectional From Origin of Replication
• DNA replication is bidirectional.
– Bidirectional replication involves two replication
forks, which move in opposite directions.
Features of DNA Replication Replication Activities Occur at Replication Folk
Features of DNA Replication Bidirectional
Features of DNA Replication Semi-discontinuous
• Replication is semi-discontinuous.
– Continuous in one strand (leading strand).
– Leading strand is formed continuously
– Discontinuous in the other strand (lagging strand).
– Lagging strand is formed discontinuously from short
DNA fragments known as Okazaki fragments.
» Involves short fragments from many RNA primers.
» Discovered by Tuneko and Reiji "O“.
– Okazaki fragments on the lagging strand must be joined to form
a continuous strand using a ligase.
Features of DNA Replication DNA Pol III Requires an RNA primer
• An RNA primer is required to initiate DNA
replication.
• A special primase synthesizes the required
primer.
• DNA Pol I excises the primer after DNA
synthesis.
DNA Replication The Primer
DNA Replication The Primer
Mechanism of DNA Replication Initiation of Replication in E. coli
• The replisome is a complex molecular machine
that carries out replication of DNA. – DNA Pol III holoenzyme, helicase and primase interact with
each other to form replisome.
– A DNA synthesis factory with the activity of each protein is
highly coordinated.
• Replisome consists of the following: – DNA-unwinding proteins.
– The priming complex (primosome).
– Two equivalents of DNA polymerase III holoenzyme.
• Initiation process: – DnaA protein binds repeats in ori, initiating DNA separation.
– DnaB, a helicase delivered by DnaC, further unwinds.
– Primase then binds and constructs the RNA primer.
Mechanism of DNA Replication Initiation of Replication in E. coli
Mechanism of DNA Replication Initiation of Replication in E. coli: Replisome
Mechanism of DNA Replication Elongation in E. coli
• Elongation: – DnaB helicase unwinds DNA.
– SSB (Single Strand Binding) protein binds to keep
strands separated.
– DNA polymerase activity on both DNA strands.
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• The junction between the newly separated
template strands and the unreplicated duplex
DNA.
• Various enzymes participate at the replication
fork to extend DNA together with a range of
DNA polymerases. – Ligases.
– Gyrase/ Topoisomerase.
– Primases.
– SSB.
– Helicases.
– Exonucleases.
Mechanism of DNA Replication Elongation: Replication Fork
Mechanism of DNA Replication Both Leading & Lagging Strand Synthesized at Replication Fork
Leading strand
Lagging strand
Okazaki fragment
Replication fork
• Gyrases
(Topoisomerases). – Remove supercoils
produced by DNA
unwinding at the
replication fork.
Mechanism of DNA Replication Elongation: Replication Fork
• DNA helicases unwind the double helix in
advance of the replication fork.
Mechanism of DNA Replication Elongation: Replication Fork
Mechanism of DNA Replication Elongation: Replication Fork
• Single-stranded binding proteins (SSBs)
stabilize single-stranded DNA. – Cooperatively bind ssDNA in a sequence-independent
manner using electrostatic interactions.
• At the replication, the leading strand and
lagging strand are synthesized simultaneously.
• The Trumbone model. – Explains how the anti-parallel template strands are
copied/replicated toward the replication fork by the
polymerase.
• To coordinate the replication of both strands,
multiple DNA Polymerases function at the
replication fork linked to clamp sliding proteins.
– DNA Pol III holoenzyme is such an example. – Mechanism discussed here in detail.
Mechanism of DNA Replication Elongation: Replication Fork
• Structure of DNA Pol III holoenzyme and
functions at the replication fork.
Mechanism of DNA Replication Elongation: Replication Fork
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Mechanism of DNA Replication Elongation: Synthesis of Okazaki Fragments
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Mechanism of DNA Replication Elongation: Synthesis of Okazaki Fragments
• DNA ligase seals the gaps between Okazaki
fragments forming a phosphodiester bond. – Discontinuous strand becomes continuous.
Mechanism of DNA Replication Elongation: Okazaki Fragments
Mechanism of DNA Replication Termination in E. coli
• Termination: – The "ter" locus signals the end of replication.
– The ter locus rich in Gs and Ts.
– A Ter protein is also involved. – The ter protein is a contrahelicase and prevents DNA
unwinding.
• Topoisomerase II (DNA gyrase) relieves
supercoiling that remains.
• Type II
topoisomerases
separate daughter
DNA molecules.
– Topoisomerase II
catalyze the
decatenation of
replication products.
Mechanism of DNA Replication Elongation & Termination in E. coli
Eukaryotic DNA Replication • Process similar to that in E. coli but more
complex.
– Eukaryotic chromosome are replicated exactly
once per cell cycle (in the S-phase of cell cycle),
which is critical for these organisms.
• Involves multiple origins of replication.
– Usually 1 Ori C per 3- 300 kbp.
• Several eukaryotic DNA polymerases exist
and differ in structure and activity complement.
– DNA polymerase alpha.
– DNA polymerase beta.
– DNA polymerase gamma.
– DNA polymerase epsilon.
Eukaryotic DNA Polymerases Summary of Structure & Functions
DNA Synthesis by Reverse
Transcription • Reverse transcriptase (RT).
– An RNA-directed DNA polymerase.
• Requires a primer.
– Transcribes RNA template into a complementary
DNA known as cDNA.
– Product is a DNA:RNA hybrid.
• Reverse transcriptase II.
– Has three enzyme activities.
– RNA-directed DNA polymerase activity.
– RNase H activity that degrades RNA in the DNA:RNA hybrids.
– DNA-directed DNA polymerase, which makes a DNA duplex
after RNase H activity destroys the RNA template.
DNA Repair
DNA Repair
• DNA repair a fundamental difference from
other molecules of life.
– RNA, protein, lipid, etc.
– All these other molecules of life can be replaced.
– But DNA must be preserved.
• Cells require a means for repair of missing,
altered or incorrect bases and mutations.
• Two principal mechanisms of DNA repair:
– Mismatch repair.
– Methods for reversing chemical damage.
DNA Repair Mismatch Repair
• Mismatch repair systems.
– Scan DNA duplexes for mismatched bases.
– Excise (exonuclease) the mispaired region and
replace (ligase) it.
• The Methyl-directed pathway of E. coli is
example.
– Since methylation occurs post-replication, repair
proteins identify methylated strand as parent,
remove mismatched bases on other strand and
replace them using base pairing.
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DNA Repair Reversing Chemical Damage
1. Pyrimidine dimers can be repaired by the
enzyme photolyase.
2. Excision repair (Base Excision Repair): – Involves the enzyme DNA glycosylase.
– DNA glycosylase removes damaged base, creating
an "AP site" (Apurinic/apyrimidinic site). – Also known as an abasic site, is a location in nucleotide, usually
DNA that has neither a purine nor a pyrimidine base, either
spontaneously or due to DNA damage.
– The enzyme AP endonuclease cleaves DNA
backbone.
– An exonuclease removes several residues.
– The and gap is repaired by DNA polymerase and
DNA ligase.
END
