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Molecular Biology
Replication

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DNA is Reproduced by Semiconservative

Replication

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DNA as Template (1 of 2)

• DNA strands serve as template

– Arrangement and nature of nitrogenous bases allow
DNA strands to serve as templates
– Complementarity of DNA strands allows each strand
to serve as template for synthesis of the other

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DNA as Template (2 of 2)

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Three Modes of Replication (1 of 2)

• Three modes of DNA replication

– Semiconservative
▪ Each replicated DNA molecule consists of one
“old” and one “new” strand
– Conservative
▪ Two newly synthesized strands come together—
original helix is conserved
– Dispersive
▪ Parental strands are dispersed into two new
double helices

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Three Modes of Replication (2 of 2)

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Meselson–Stahl Experiment (1 of 2)

• Meselson and Stahl (1958)

– 15N-labeled E. coli grown in medium containing 14N
– Each new DNA molecule consists of one old and one
newly synthesized strand
– Provided strong evidence that DNA is
semiconservative in prokaryotes

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Meselson–Stahl Experiment (2 of 2)

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Semiconservative DNA in Eukaryotes

• Taylor–Woods–Hughes (1957)
– Vicia faba (broad bean) was used to demonstrate
DNA replication is semiconservative in
eukaryotes
– Monitored process of replication with labeled
3H-thymidine and performed autoradiography

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Autoradiography
• Autoradiography
– Pinpoints location of radioisotope in cell
– Photographic emulsion placed over cellular material
and stored in the dark
– Develops much like photographic film
– Result: Presence of dark grains identifies location of
newly synthesized DNA

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Replication of a Single Chromosome

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Origins, Forks, and Units of Replication (1 of

2)

• DNA replication
– DNA replication begins at the ORI (origin of
replication)
– At site of replication, helix is unwound, creating
replication fork
– Replication is bidirectional; therefore, there are two
replication forks
– Replicon: Length of DNA replicated

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Origins, Forks, and Units of Replication (2 of

2)

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Bacterial Replication
• Bacteria have only one ORI
– Single circular DNA
– DNA synthesis originates at OriC
– E. coli replicon consists of entire genome
▪ 4.6 million base pairs

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DNA Synthesis in Bacteria Involves Five

Polymerases, as Well as Other Enzymes

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DNA Synthesis in Bacteria

• DNA synthesis in bacteria involves five polymerases
(DNA Pol)
– DNA polymerase I
– DNA polymerase II
– DNA polymerase III
– DNA polymerase IV
– DNA polymerase V

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DNA Polymerase I (1 of 2)

• DNA polymerase I
– Isolated enzyme from E. coli
– Enzyme directs DNA synthesis
– Requires DNA template and all four
deoxyribonucleoside triphosphates (dNTPs)
– Enzyme consists of polypeptide with 928 amino acids

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DNA Polymerase I (2 of 2)

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Chain Elongation (1 of 2)

• Chain elongation by DNA polymerase I

– Occurs in 5 to 3 direction by adding one nucleotide
at a time to 3 end
– Nucleotide added, two terminal phosphates cleaved
off, providing newly exposed 3-OH
– 3-OH can participate in addition of another nucleotide
as DNA synthesis proceeds

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Chain Elongation (2 of 2)

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DNA Pol I, II, and III (1 of 2)

• DNA Pol I, II, and III

– Can elongate existing DNA strand (primer)
– Cannot initiate DNA synthesis
• Exonuclease activity 3–5
– All three possess 3' to 5' exonuclease activity:
proofread newly synthesized DNA, remove/replace
incorrect nucleotides
• Exonuclease activity 5–3
– Only DNA polymerase I
– Excises primers—fills in gaps left behind

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DNA Pol I, II, and III (2 of 2)

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DNA Repair

• DNA polymerases I, II, IV, and V

– Involved in various aspects of DNA repair
– Repair DNA damaged by external forces such as UV
light

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DNA Pol III Holoenzyme (1 of 3)

• DNA polymerase III
– Holoenzyme: Active form of DNA Pol III
– Holoenzyme contains core enzyme complexes made
up of subunits
– Subunits each have separate functions
▪  – 5–3 polymerization
▪  – 3–5 exonuclease
▪  – Core assembly

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DNA Pol III Holoenzyme (2 of 3)

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DNA Pol III Holoenzyme (3 of 3)

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Many Complex Issues Must Be Resolved

during DNA Replication

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DNA Replication Issues (1 of 4)

• Seven key issues must be resolved during DNA
replication:
1. Unwinding of helix
2. Reduce increased coiling generated during
unwinding
3. Synthesis of primer for initiation
4. Discontinuous synthesis of second strand
5. Removal of the RNA primers
6. Joining of gap-filling DNA to adjacent strand
7. Proofreading

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Summary of DNA Synthesis

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DnaA—Unwinding the Helix

• DnaA
– Initiator protein encoded by dnaA gene
– Binds to ORI causing conformation change
– Causes helix to destabilize and open up
– Exposes ssDNA

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DNA Helicase

• DNA helicase
– Made of DnaB polypeptides
– Hexamer of subunits: Assembles around exposed
ssDNA
– Subsequently recruits holoenzyme to bind
replication fork and initiate replication
– Helicases require energy supplied by hydrolysis of
ATP—denatures hydrogen bonds and stabilizes
double helix

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SSBPs
• Single-stranded binding proteins (SSBPs)
– Stabilize the open conformation of helix
– Bind specifically to single strands of DNA

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Supercoiling

• DNA gyrase
– Enzyme relieves coiled tension from unwinding of
helix (DNA supercoiling)
– Member of larger enzyme group: DNA
topoisomerases
– Makes single- or double-stranded cuts
– Driven by energy released during ATP hydrolysis

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RNA Polymerase: Primase

• Primase: RNA polymerase
– Recruited to replication form by helicase
– Synthesizes RNA primer
– Provides free 3-OH required by DNA polymerase III
for elongation

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DNA Replication Issues (2 of 4)

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RNA Priming
• DNA polymerase I
– Removes primer and replaces it with DNA
• RNA priming
– Universal phenomenon
– Found in bacteria, viruses, and several eukaryotic
organisms

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Continuous and Discontinuous DNA

Synthesis

• Two strands of double helix are antiparallel: 5–3 and

3–5
– DNA Pol III ONLY synthesizes 5–3
• Continuous DNA synthesis
– Leading strand
• Discontinuous DNA synthesis
– Lagging strand

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DNA Replication Issues (3 of 4)

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Okazaki Fragments

• Okazaki fragments
– Lagging strand synthesized as Okazaki fragments,
each with RNA primer
• DNA polymerase I
– Removes primers on lagging strand
– DNA ligase
▪ Catalyzes formation of phosphodiester bonds
▪ Seals nicks and joins fragments

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Concurrent Synthesis
• Both DNA strands synthesized concurrently
– Concurrent DNA synthesis achieved on both strands
at single replication fork
– Lagging strand is looped
– Inverts physical but not biochemical direction
– DNA clamp prevents core enzyme dissociation from
template

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DNA Replication Issues (4 of 4)

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Proofreading
• Proofreading and error correction
– Integral part of DNA replication
– DNA polymerase not always perfect
– Synthesis of noncomplementary base pairs inserted
occasionally
– DNA polymerase exonuclease activity of 3–5 allows
for excise of nucleotides

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A Coherent Model Summarizes DNA

Replication

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Summarizing It All…
• Enzymes and proteins are essential to DNA synthesis
– DNA polymerase III core enzymes
– SSBPs: single-stranded binding proteins
– DNA gyrase
– DNA helicase
– RNA primers

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Summary of DNA Synthesis

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Replication is Controlled by a Variety of

Genes

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Replication Controlled by Variety of Genes

(1 of 2)

• Mutations
– Interrupt or impair aspects of replication
– Examples: Lethal mutations
▪ Ligase-deficient mutations
▪ Proofreading-deficient mutations
– Conditional mutations: Expressed under specific
conditions
– Table 11.4: E. coli genes and their roles

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Replication Controlled by Variety of Genes

(2 of 2)

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Temperature-Sensitive Mutation

• Temperature-sensitive mutation
– Example of conditional mutation
– May not be expressed at particular permissive
temperature
– Mutant cells grown at restrictive temperature and
mutant phenotype expressed

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Eukaryotic DNA Replication is Similar

to Replication in Bacteria, but is More
Complex

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Eukaryotic DNA Replication

• Eukaryotic and bacterial DNA replication shares

many features
– Double-stranded DNA unwound at ORI
– Replication forks formed
– Bidirectional synthesis creates leading and lagging
strands
– Eukaryotic polymerases require four
deoxyribonucleoside triphosphates, template, and
primer

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Eukaryotic—More Complex
• Eukaryotic DNA replication is more complex
– More DNA than prokaryotic cells
– Linear chromosomes
– DNA complexed with nucleosomes

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Eukaryotes—Multiple ORIs (1 of 2)

• Eukaryotic replication: Multiple ORIs

– Eukaryotic chromosomes contain multiple ORIs
– Facilitates rapid synthesis of large quantity of DNA

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Eukaryotes—Multiple ORIs (2 of 2)

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ARSs

• Yeast genomes contain 250–400 origins

• Yeast ORI:
– Autonomously replicating sequences (ARSs)
– 120 base pairs of consensus sequences
– Consensus sequence: Sequence that is the same in
all yeast ARSs

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Prereplication Complex
• Eukaryotic ORIs control timing of DNA replication
• Prereplication complex (pre-RC)
– Assembles at replication ORIs
– Early G1 phase of cell cycle:
▪ Origin recognition complex (ORC) recognizes
ORI and tags ORI as site of initiation

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Multiple Eukaryotic DNA Polymerases (1 of

2)

• DNA polymerases involved in nuclear genome DNA

replication
– Pol , , and : Involved in initiation and elongation
– Pol 
▪ Possesses low processivity
▪ RNA primer synthesis during initiation on leading
and lagging strands
• Table 11.5: Others involved in repair processes

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Multiple Eukaryotic DNA Polymerases (2 of

2)

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Polymerase Switching

• Polymerase switching occurs once the primer is in

place
– Pol  and  replaced by Pol  for elongation
– Pol  synthesizes lagging strand
– Pol  synthesizes leading strand

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Replication through Chromatin (1 of 2)

• Eukaryotic DNA complexed with binding proteins
(chromatin)
• 200 base pair nucleosomes wrap around eight histone
proteins
– Nucleosomes must be stripped away before
polymerase can begin synthesis

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Replication through Chromatin (2 of 2)

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Telomeres Solve Stability and

Replication Problems at Eukaryotic
Chromosome Ends

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Telomeres (1 of 2)

• Telomeres
– Inert chromosomal ends that protect intact eukaryotic
chromosomes from improper fusion or degradation
– Long stretches of short repeating sequences preserve
the integrity/stability of chromosomes
• Telomere t-loops and a complex of six proteins binds
and stabilizes chromosome ends
– Forms the shelterin complex

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Telomeres (2 of 2)

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Telomerase (1 of 2)

• Telomerase
– Eukaryotic enzyme
– Ribonucleoprotein: RNA serves as template for
synthesis of DNA complement
1. Telomerase RNA component (TERC)
2. Telomerase reverse transcriptase (TERT)
– Once RNA primer removed on lagging strand, no free
3-OH to elongate
– Telomerase adds repeats of six-nucleotide sequence
to 3 end to fill gaps

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Telomerase (2 of 2)

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Telomerase Activity

• Telomeres of chromosomes shorten with each cell

division
– In most eukaryotic somatic cells, telomerase is not
active
• Stem cells and malignant cells maintain telomerase
activity—immortalized
• Telomerase activity and telomere length linked to aging,
cancer, and other diseases

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