9/20/2010

Fundamental Genetics
Lecture 10 The Flow of Biological Information
Replication

DNA Replication DNA

and Synthesis Transcription

RNA

Translation
John Donnie A. Ramos, Ph.D.
Dept. of Biological Sciences
College of Science
University of Santo Tomas Protein

Modes of DNA Replication Semiconservative Replication

Semiconservative Replication in Prokaryotes Semiconservative Replication in Prokaryotes

Mathew Messelson and Franklin Stahl (1958)

Expected results of the Messelson-Stahl experiment

15N– heavy isotope of N (contains 1 more
neutron) compared to 14N

15N has high sedimentation rate in cesium
chloride compared to 14N

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Semiconservative Replication in Eukaryotes Replication of E. coli Plasmid

J. Herbert Taylor, Philip
Shown by John Cairns (1981) using
Woods, and Walter Hughes radioisotopes and radiography
(1957)

Replication starts in a single OriC –

Used root tip cells from Vicia origin of replication (245 bp)
faba (broad bean)

Replication is bidirectional

Monitored replication using
3H-Thymidine to label DNA
Replication fork – unwound DNA helix

Replicon – replicated DNA

Used autoradiography to
determine the incorporation
Ter region – region of replication
of 3H-Thymidine termination

Arrested cells at metaphase
using colchicine

DNA Synthesis in Microorganisms Chain Elongation

5’ to 3’ direction of DNA synthesis (requires 3’ end of the DNA template)

DNA polymerase I (928
Each step incorporates free 3’ OH group for further elongation
aa) – catalyses the
synthesis of DNA in vitro
(A. Kornberg, 1957)

Requirements:

Deoxyribonucleoside
triphosphates, dNTPs
(dATP, dCTP, dGTP, dTTP)

DNA template

Primer

DNA replication using DNA polymerase is of high fidelity (highly

accurate)

With exonuclease activity (proofreading ability)

DNA Polymerases Replication in Prokaryotes

All 3 types requires a primer
1. Unwinding of DNA helix

Complex proteins (100,000 Da)
2. Initiation of DNA synthesis
Functions of DNA polymerases
in vivo 3. DNA synthesis proper (elongation)

DNA Pol I – proofreading; 4. Sealing gaps
removes primers and fills gaps
5. Proofreading and error correction

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.

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Unwinding of DNA Helix Initiation of DNA Synthesis

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

Binding of Dna B and Dna C to the DNA template
unwound helix
Catalysed by primase (an RNA polymerase)

Single-stranded binding proteins
Pimase does not require free 3’ end to initiate synthesis (not unlike
(SSBPs) – prevents reannealing of DNA polymerase III)
replication bubble.

Function of primase will be continued by DNA polymerase III.

DNA gyrase (a DNA topoisomerase)
– relaxes the supercoiling of DNA
helix

Sealing of Gaps, Proofreading

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
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

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 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 Telomerase

Enzyme that adds TTGGGG
repeats on the telomeres (first

Telomeres – linear ends of identified in Tetrahymena)
eukaryotic chromosomes

Prevents shortening of

Problem with lagging chromosomes
strand: no 3’ needed by
Forms a “hairpin loop” on
DNA polymerase I (after chromosome ends using G-G
removal of RNA primers) bonds

Possible result: chromosome
Creates a free 3’ on lagging
strand that can be used by
with shorter lagging strand
DNA polymerase I to replaced
every replication step 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

Homologous Gene Conversion

Recombination

Exchange of genetic information between non-homologous

Exchange of genetic material chromosomes (non-reciprocal genetic exchange)

Directed by specific
Type of chromosome mutation (recombination)
enzymes:
First identified in Neurospora (by Mary Mitchell)

Endonuclease – introduces
Can be repaired but forms recombined genetic material
single strand nicks

Ligase – seals loose ends
(nicks)

Rec A protein promotes the

exchange of reciprocal
single-stranded DNA
molecules and it enhances
hydrogen bond formation
during strand displacement