Fundamental Genetics

Lecture 10

DNA Replication
and Synthesis
John Donnie A. Ramos, Ph.D.
Dept. of Biological Sciences
College of Science
University of Santo Tomas

The Flow of Biological Information

Replication

DNA

Transcription

RNA

Translation

Protein

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 Modes of DNA Replication

Semiconservative Replication

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Semiconservative Replication in Prokaryotes
‰ Mathew Messelson and Franklin Stahl (1958)
‰ 15N– heavy isotope of N (contains 1 more
neutron) compared to 14N
‰ 15N has high sedimentation rate in cesium
chloride compared to 14N

Semiconservative Replication in Prokaryotes

‰ Expected results of the Messelson-Stahl experiment

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 Semiconservative Replication in Eukaryotes
‰ J. Herbert Taylor, Philip
Woods, and Walter Hughes
(1957)
‰ Used root tip cells from Vicia
faba (broad bean)
‰ Monitored replication using
3H-Thymidine to label DNA

‰ Used autoradiography to
determine the incorporation
of 3H-Thymidine
‰ Arrested cells at metaphase
using colchicine

Replication of E. coli Plasmid

‰ Shown by John Cairns (1981) using
radioisotopes and radiography
‰ Replication starts in a single OriC –
origin of replication (245 bp)
‰ Replication is bidirectional
‰ Replication fork – unwound DNA helix
‰ Replicon – replicated DNA
‰ Ter region – region of replication
termination

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 DNA Synthesis in Microorganisms
‰ DNA polymerase I (928 aa) – catalyses the synthesis of DNA in vitro
(A. Kornberg, 1957)
‰ Requirements:
‰ Deoxyribonucleoside triphosphates, dNTPs (dATP, dCTP, dGTP, dTTP)
‰ DNA template
‰ Primer

Chain Elongation
‰ 5’ to 3’ direction of DNA synthesis (requires 3’ end of the DNA template)
‰ Each step incorporates free 3’ OH group for further elongation

‰ DNA replication using DNA polymerase is of high fidelity (highly

accurate)
‰ With exonuclease activity (proofreading ability)

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 DNA Polymerases
‰ All 3 types requires a primer
‰ Complex proteins (100,000 Da)
Functions of DNA polymerases
in vivo
‰ DNA Pol I – proofreading;
removes primers and fills gaps
‰ DNA Pol II - mainly involved in
DNA repair from external
damage
‰ DNA Pol III – main enzyme
involved in DNA synthesis
‰ a holoenzyme (>600,000 Da)
– forms replisome when
attached to a replication fork.

Replication in Prokaryotes
1. Unwinding of DNA helix
2. Initiation of DNA synthesis
3. DNA synthesis proper (elongation)
4. Sealing gaps
5. Proofreading and error correction

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 Unwinding of DNA Helix
‰ Takes place in oriC (245 bp) –
repeating 9mers and 13mers
‰ Function of helicases (Dna A, B, C)
– requires ATP hydrolysis to break
hydrogen bonds
‰ Initiated by Dna A – binds to 9mers
‰ Binding of Dna B and Dna C to
unwound helix
‰ Single-stranded binding proteins
(SSBPs) – prevents reannealing of
replication bubble.
‰ DNA gyrase (a DNA topoisomerase)
– relaxes the supercoiling of DNA
helix

Initiation of DNA Synthesis

‰ Synthesis of RNA primer – 5 to 15 RNA bases complementary to

the DNA template
‰ Catalysed by primase (an RNA polymerase)
‰ Pimase does not require free 3’ end to initiate synthesis (not unlike
DNA polymerase III)
‰ Function of primase will be continued by DNA polymerase III.

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 DNA Synthesis (Elongation)
‰ Function of DNA polymerase III
‰ Requires free 3’ end
‰ Direction of elongation: 5’ to 3’
‰ DNA synthesis is continuous in 3’ to 5’
DNA strand (leading strand) and
discontinuous in the 5’ to 3’ DNA strand
(lagging strand).
‰ Okazaki fragments – short DNA
fragments produced in the lagging
strand
‰ Concurrent synthesis of leading and
lagging strands occur by using DNA pol
dimer and by a looping mechanism for
the lagging strand

Sealing of Gaps, Proofreading

and Error Correction
‰ DNA polymerase I removes all RNA bases produced
by primase (creates gaps in the lagging strand) and
replaces it with DNA bases (U to T).
‰ DNA ligase seals the gaps by forming
phosphodiester bonds
‰ Exonuclease proofreading (identification of
mismatched bases) is a function of both DNA
polemerase I and III (both with 3’-5’ exonuclease
activity)
‰ ε subunit of DNA polymerase III is involved in
proofreading.
‰ Assures high fidelity of DNA replication

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 Mutations Affect Replication

Replication in Eukaryotes
‰ Presence of multiple replication origin
(faster replication, guarantees replication
of a big genome) – 25K replicons in
mammalian cells
‰ Autonomously replicating sequences
(ARSs) – origin of replication in yeasts
(11 bp)
‰ Origin site is AT rich region
‰ Helicase unwinds double stranded DNA
and removes histone proteins from DNA
‰ Histones reassociates while DNA
synthesis occurs.

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 Eukaryotic DNA Polymerases
‰ Pol α - initiates nuclear DNA synthesis
‰ 4 subunits (2 acts primase – produces RNA primers)
‰ Acts on both leading and lagging strands
‰ 2 other subunits continue elongation step (DNA synthesis)
‰ Low processivity (short length of synthesized DNA prior to dissociation)
‰ Pol δ - replaces Pol α (called polymerase switching)
‰ High processivity (during elongation)
‰ With 3’-5’ exonuclease activity (proofreading)
‰ Pol ε - nuclear DNA synthesis
‰ Pol β - DNA repair (the only eukaryotic DNA polymerase with single
subunit)
‰ Pol ξ - DNA repair
‰ Pol γ - mitochondrial DNA synthesis (encoded by nuclear gene)

‰ Eukaryotes has a high copy number of DNA polymerases (ex. Pol α

may be up to 50K copies)

Eukaryotic DNA Replication

‰ Telomeres – linear ends of
eukaryotic chromosomes
‰ Problem with lagging
strand: no 3’ needed by
DNA polymerase I (after
removal of RNA primers)
‰ Possible result:
chromosome with shorter
lagging strand every
replication step

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 Telomerase
‰ Enzyme that adds TTGGGG
repeats on the telomeres (first
identified in Tetrahymena)
‰ Prevents shortening of
chromosomes
‰ Forms a “hairpin loop” on
chromosome ends using G-G
bonds
‰ Creates a free 3’ on lagging
strand that can be used by
DNA polymerase I to replaced
the removed RNA primer
‰ Telomerase is a
ribonucleoprotein and contains
RNA sequence (5’ AACCCC 3”-
serving as template) – reverse
transcriptase
‰ Cleavage of loop after DNA
synthesis

DNA
Recombination
‰ Exchange of genetic
material
‰ Homologous
recombination
‰ Ex. Rec A protein
(produces recB, recC
and recD genes)

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 Gene Conversion
‰ Exchange of genetic information between non-homologous
chromosomes
‰ Type of chromosome mutation (recombination)
‰ First identified in Neurospora (by Mary Mitchell)
‰ Can be repaired but forms recombined genetic material

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