The Central Dogma
DNA structure and DNA replication
DNA replication (continued)
RNA Synthesis
Protein synthesis
DNA – an emblem of the 20th century.
1.! A simple yet elegant structure – a double
helix with a sugar phosphate “backbone”
linked to 4 types of nucleotide on the
inside that are paired according to basic
rules. Amazingly this simple molecule has
the capacity to specify Earth’s incredible
biological diversity.
2.! The double-stranded structure suggests
a mode of copying (replication)
3.! The “strings” of the 4 bases are a digital
code that specifies life.
Prof. David McConnell
Smurfit Institute of Genetics
These lectures cover the research that led to the
elucidation of the replication (copying/ reproduction) of
DNA, and how DNA can generate protein products.
Summary of Lecture 1
1.! DNA is a double stranded helix (Watson and Crick 1957)
2.! The strands are anti-parallel: 5’ to 3’ and 3’ to 5’
3.! The two strands are held together by base pairs: A=T and G=C.
4.! The strands have complementary base sequences.
5.! The structure of DNA is independent of base sequence (not quite
true)
6.! DNA replication is semi-conservative (Meselson-Stahl, 1958)
7.! So DNA must unwind when it is replicating
8.! Replication of the Escherichia coli genome (a single circular DNA)
starts at a specific site (ori) and is bi-directional (Cairns, 1963).
Replication as a Process
1. Double-stranded DNA must
unwind.!
2. The junction of the unwound !
molecules is a replication fork.!
3. A new strand is formed by pairing !
complementary bases with the!
old strand.!
4. Two molecules are made. !
Each has one new and one old !
DNA strand. !
DNA Replication is Semi-discontinuous!
Continuous synthesis!
Lecture 2 Outline
1.! There are many enzymes involved in DNA replication
2.! The main replicative enzyme is DNA polymerase III
3.! The enzyme is composed of several proteins
4." RNA primers are required for replication.
5.! Additional features of the replication process.
Discontinuous synthesis!
Arthur Kornberg (1957)
Kornberg devised an in vitro assay!
Protein extract from E. coli!
+
!template DNA!
+
!substrates!
He guessed these would be:!
dATP; dTTP; dGTP and dCTP!
Set out to identify and purify !
an enzyme that could make DNA!
He guessed that Mg2+ would be !
required!
Discovered DNA polymerase I!
Arthur Kornberg (1957)
He found he could to make DNA !
in the test tube (in vitro)!
Called the enzyme DNA polymerase!
He purified the DNA polymerase!
He guessd that ATP would be !
Needed as an energy source. Not so!
Kornberg used the in vitro assay to characterize!
the DNA polymerizing activity!
- bases are ONLY added to the 3# end of newly !
replicating DNA!
5#!
3#!
3#!
5#!
3#!
5#!
3#!
3#!
5#
Template!
5#
Template!
5#
Template!
3#!
Found to be a single polypeptide!
-therefore DNA synthesis occurs only in the!
5# to 3# direction!
928 amino acids long!
DNAP I could only add bases to a primer
Kornberg discovered that DNA polymerase worked!
much better on single than double stranded DNA.!
DNA with short single stranded regions was a good !
template.!
5#!
3#!
5#!
3#!
5#!
3#!
3#!
3#!
5#
Not a template!
5#
Good template!
3#!
5#
Good template!
Kornberg discovered that DNA polymerase I could !
not start a new DNA strand.!
It could only extend a strand (the primer)that was !
base paired with a template.!
3#!
5#!
3#!
5#!
3#!
3#!
5#
No reaction!
5#
Good template!
3#!
5#
Good template!
THERE WAS A LARGE CONCEPTUAL PROBLEM!
Proposal: the other strand is replicated !
“backwards and discontinously”!
Consider one replication fork!
3#!
3#!
3#!
5#!
Primer!
3#!
5#!
Primer!
Continuous replication!
Continuous replication!
5#!
5#!
Direction of unwinding
Direction of unwinding
3#!
How is the other strand replicated?!
3#!
er!
5#! Prim
3#!
Discontinuous replication!
er!
5#! Prim
3#!
5#!
5#!
Leading and lagging strands!
3#!
Leading strand!
3#!
5#!
Primer!
Continuous replication!
5#!
Evidence for the Semi-Discontinuous replication !
model was provided by Okazaki (1968)!
Direction of unwinding
er!
3#!
5#! Prim
3#!
Discontinuous replication!
er!
5#! Prim
3#!
Lagging strand!
5#!
Evidence for Semi-Discontinuous Replication!
Evidence for Semi-Discontinuous Replication!
Pulse-chase experiment !
Pulse-chase experiment !
Bacterial!
culture!
Bacteria are!
replicating!
Time zero. !
Add 3H Thymidine (T)!
For a SHORT time!
(i.e. seconds)!
The pulse!
Flood with !
non-radioactive T!
Allow replication!
to continue !
The chase!
Purify DNA at different times
Denature and measure size of all radioactive material
Pulse with 3H Thymidine!
A few seconds!
DNA is radioactive!
Radioactivity will only!
be in the DNA that was !
made during the pulse!
Flood the culture with non-radioactive T!
Replication continues!
Harvest the bacteria!
at different times!
Purify the DNA!
Separate the strands!
(using alkali conditions)!
Centrifuge the single stranded DNA!
Evidence for Semi-Discontinuous Replication!
Pulse-chase experiment !
Results of pulse-chase experiment: after the pulse !
3’
5’
Chase!
Leading strand
Centrifuge tube
Large molecule
Contains aqueous solution
Layer the single stranded DNA sample on top
Centrifuge
Pulse
5’
3’
Small molecules
Pierce the tube on the bottom
Collect drops from the tube
Measure the radioactivity in each drop
5’
Lagging strand
Plot radioactivity per drop
Evidence for Semi-Discontinuous Replication!
Pulse-chase experiment!
Results of pulse-chase experiment: after the chase !
3’
5’
Chase!
Leading strand
See small and large DNA just after the pulse !
Large molecule
DNA purified just after the pulse
Small
Shows some very large molecules
the leading strand
5’
Pulse and chase
3’
Large molecule
And some very small ones
Large
the fragments from the lagging
strand
Evidence for Semi-Discontinuous Replication!
Pulse-chase experiment!
See only large DNA after a long chase !
Lagging strand
5’
DNA replication is semi-discontinuous
Continuous synthesis!
DNA purified just after long chase
Shows only very large molecules
the leading strand
the fragments from the lagging
strand have been joined together
Discontinuous synthesis!
Features of DNA Replication
The enzymology of DNA polymerase I
•! DNA replication is semiconservative
–! Each strand of template DNA is being copied.
•! DNA replication is bidirectional
–! Bidirectional replication involves two replication forks, which move
in opposite directions
•! DNA Polymerase I has THREE different
enzymatic activities in a single polypeptide
•! the 5’ to 3’ DNA polymerizing activity
•! DNA replication is semidiscontinuous
–! The leading strand copies continuously
–! The lagging strand copies in segments (Okazaki fragments) which
must be joined
•! a 3’ to 5’ exonuclease activity
•! a 5’ to 3’ exonuclease activity
DNA SYNTHESIS REACTION
The 5’ to 3’ DNA polymerizing activity
P
5’
3’
P
CH2
Base
O
CH2
P
5’
The hydrolysis of the !
phosphodiester bond!
energises the reaction.!
Nucleotides are added at the 3'-end of the new strand
Base
O
P
CH2
3’
5' end of strand
O
CH2
Base
Base
O
3'
P
Synthesis reaction
P
5' CH2
3'
P
P
OH
P
products
H 20
+
O
CH2
O
P
Base
Base
OH
3' end of strand
OH
Proof reading activity
of the 3’ to 5’ exonuclease.
Why the exonuclease activities?
•! The 3'-5' exonuclease activity
serves a proofreading function
•! It removes incorrectly matched
bases, so that the polymerase can
try again.
DNAP I stalls if the incorrect
base is added - it cannot add the
next base in the chain
Proof reading activity is slow
compared to polymerizing
activity, but the stalling of
DNAP I after insertion of an
incorrect base allows the
proofreading activity to
catch up with the polymerizing
activity and remove the
incorrect base.
DNA Replication is accurate
(In E. coli: 1 error/109 -1010 bases added)
Why the 5’-3’ exonuclease activity?
What ensures that it is so accurate?"
1) Base-pairing specificity at the active site"
-!correct geometry in the active site occurs only with correctly
paired bases BUT the wrong base still gets inserted 1/ 104
-105 bases added"
•! The 5’-3' exonuclease activity is used to
excise RNA primers in a reaction called “nick
translation”
2) Proofreading activity by 3#-5# exonuclease"
- removes mispaired bases from 3# end of DNA"
-!increases the accuracy of replication 102 -103 fold"
•! Describe the role of this later
3) Mismatch repair system"
- corrects mismatches AFTER DNA replication"
Is DNA Polymerase I the principal replication
enzyme?
In 1969 John Cairns and Paula deLucia isolated a
mutant E. coli strain with only 1% DNAP I activity
(polA)
Other clues….
-! DNAP I is slow (600 bases added/minute – would
take 100 hrs to replicate genome instead of 40
minutes)
- mutant was super sensitive to UV radiation
- but otherwise the mutant was fine i.e. it could divide,
so obviously it could replicate its DNA
- DNAP I is only moderately processive
(processivity refers to the number of bases added to
a growing DNA chain before the enzyme dissociates
from the template)
Inference:
•! DNAP I may NOT BE the principal replication
enzyme in E. coli
Inference:
•! There might be additional DNA polymerases.
•! Sought other polymerases in the polA mutant
So if it is not the chief replication
enzyme then what does DNAP I do?
The DNA Polymerase Family
A total of 5 different DNAPs have been
discovered in E. coli
-! functions in multiple processes that require
only short lengths of DNA synthesis
-! has a major role in DNA repair (CairnsdeLucia mutant was UV-sensitive)
-! its role in DNA replication is to remove primers
and fill in the gaps left behind
- for this it needs the nick-translation activity
•!
•!
•!
•!
•!
DNAP I: functions in repair and replication
DNAP II: functions in DNA repair (proven in 1999)
DNAP III: principal DNA replication enzyme
DNAP IV: functions in DNA repair (discovered in 1999)
DNAP V: functions in DNA repair (discovered in 1999)
DNA SYNTHESIS REACTION
DNA Polymerase III
P
P
CH2
The "real" replicative polymerase in E. coli
Base
O
5' end of strand
CH2
Base
O
•! It is fast: up to 1,000 bases added/sec/enzyme
P
P
CH2
•! It is highly processive: >500,000 bases added before
dissociating
CH2
Base
O
Synthesis reaction
P
5' CH2
P
P
OH
P
products
H 20
+
3'
P
•! It is accurate: makes 1 error in 107 bases added; with
proofreading, this gives a final error rate of 1 in 1010
overall.
O
Base
O
CH2
O
P
Base
Base
3'
OH
3' end of strand
OH
DNA must be “Primed” before DNA Polymerase can replicate
The subunits of E. coli DNA polymerase III!
Subunit! Function!
Holoenzyme!
Core!
Enzyme!
dimer!
!"
#"
$"
%"
&"
'"
("
(#"
)"
*"
DNA polymerase cannot initiate polymerisation
5# to 3# polymerizing activity!
3# to 5# exonuclease activity!
! and # assembly (scaffold)!
Assembly of holoenzyme on DNA!
Sliding clamp = processivity factor!
Clamp-loading complex!
Clamp-loading complex!
Clamp-loading complex!
Clamp-loading complex!
Clamp-loading complex!
de novo on double stranded DNA
Okazaki and colleagues provided evidence for short
stretches of RNA linked to nascent chains of DNA
during replication.
These RNA segments are called “primers”
1.! Sugino et al., (1972) isolated Okazaki fragments from E. coli
after pulsing with 3H-U (incorporates into RNA and not DNA)
Conclusions of this and later work
and found it associated with newly replicated DNA.
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=426820&blobtype=pdf
2. In follow up experiments Sugino et al., (1973) isolated
Okazaki fragments after a short pulse (3H-dT) by banding
on a CsCl gradient.
3
4!
Treatment of the Okazaki fragments with alkali
1.! There is a covalent linkage between ribonucleotides and
deoxyribonucleotides in the newly synthesised DNA.
2.! RNA fragments (10 to 20 nt) are located at the 5’ end of the
nascent fragments and are required for priming de novo
DNA synthesis.
(hydrolyses RNA but not DNA) or ribonuclease
5.! These fragments are made by a special RNA polymerase
called RNA primase - this is resistant to the drug rifampicin
resulted in a small reduction in density.
4!
If you chop an RNA primer off the end of an Okazaki
fragment you expect the density of the fragment to be reduced
because RNA is denser than DNA.
The initiation of the leading strand is carried out by the main
E. coli RNA polymerase (which is sensitive to rifampicin) at
the origin of replication, called oriC in E. coli - see below.
1
X
Puzzles
How do the Okazaki strands become linked to each other?
Do the RNA primers stay in the new DNA?
2
3
If not how are they removed?
4
1!
DNA pol III making new Okazaki DNA (red) approaches previous
RNA primer (green) at X
DNA pol I takes over, extending new DNA (blue) and digesting RNA primer
(green) - this is called “nick translation”.
DNA pol I dissociates and DNA ligase seals the nick.
2!
3
Proteins Involved in DNA Replication
in E. coli
1
X
2
3
4
1!
2!
3
DNA pol III making new Okazaki DNA (red) approaches previous
RNA primer (green) at X
DNA pol I takes over, extending new DNA (blue) and digesting RNA primer
(green) - this is called “nick translation”.
DNA pol I dissociates and DNA ligase seals the nick.
Protein Name
Function
DNA Gyrase (Topoisomerase)
SSB
DnaA
HU
PriA
PriB
PriC
DnaB
DnaC
DnaT
Primase
DNAP III holoenzyme
DNAP I
Ligase
Tus
Unwinding DNA
Single-stranded DNA binding
Initiation factor
Histone-like (DNA binding and bending)
Primosome assembly
Primosome assembly
Primosome assembly
DNA unwinding (helicase)
DnaB chaperone
Assists DnaC in delivery of DnaB
Synthesis of an RNA primer
Elongation (DNA synthesis)
Excises RNA primer, fills in with DNA
Covalently links Okazaki fragments
Termination
Replication - role of helicase
Replication - role of DNA pol III
Overall direction
of replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
3’
5’
3’
3’
5’
Helicase: this unwinds DNA
DNA pol III adds DNA nucleotides !
to the RNA primer.!
3’
5’
5’
3’
5’
DNA pol III adds DNA nucleotides !
to the RNA primer.!
DNA polymerase proofreads bases added and !
replaces incorrect nucleotides.!
Replication: leading and lagging strands
Overall direction
of replication
Replication: leading and lagging strands 2
Overall direction
of replication
3’
3’
5’
3’
3’
5’
5’
3’
3’
5’
5’
Okazaki fragment!
5’
3’
3’ 5’
3’
5’
5’
Leading strand synthesis continues in a !
5# to 3# direction.!
Leading strand synthesis continues in a !
5# to 3# direction.!
Discontinuous synthesis produces a series of!
5# to 3# DNA segments - the Okazaki fragments. !
Replication: leading and lagging strands 3
Overall direction
of replication
Replication: leading and lagging strands 4
3’
3’
3’
5’
5’
3’
5’
Okazaki fragment!
3’
5’
3’ 5’
5’
3’
3’
5’
5’
3’ 5’
3’5’
3’
5’
Leading strand synthesis continues in a !
5# to 3# direction.!
Leading strand synthesis continues in a !
5# to 3# direction.!
Discontinuous synthesis produces 5# to 3# DNA !
segments called Okazaki fragments. !
Discontinuous synthesis produces 5# to 3# DNA !
segments called Okazaki fragments. !
Replication: leading and lagging strands 5
1
X
3’
5’
3’
2
5’
3’
5’
3’5’
3’5’
3’
3
5’
Leading strand synthesis continues in a !
5# to 3# direction.!
Discontinuous synthesis produces 5# to 3# DNA !
segments called Okazaki fragments. !
4
1!
2!
3
DNA pol III making new Okazaki DNA (red) approaches previous
RNA primer (green) at X
DNA pol I takes over, extending new DNA (blue) and digesting RNA primer
(green) - this is called “nick translation”.
DNA pol I dissociates and DNA ligase seals the nick.
DNA Synthesis
DNA REPLICATION
-!problem of going in two directions at the same time
Simultaneous replication
occurs via looping of lagging
strand
3 Pol III synthesises leading strand
1 Helicase opens helix
2
Topoisomerase
nicks DNA to
relieve tension
from unwinding
4 Primase synthesises RNA primer
5
6
Pol I excises RNA primer; fills gap
7
Pol III elongates primer;
produces Okazaki fragment
DNA ligase links Okazaki
fragments to form
continuous strand
Pol I takes over from Pol III
and runs into RNA primer
Simultaneous Replication Occurs via
Looping of the Lagging Strand
Initiation of DNA replication, but not continuation,
was shown to be sensitive to rifampicin
(an antibiotic that inhibits E. coli RNA polymerase).
How do we explain this?
The key idea is that RNA polymerase starts the
whole process of DNA replication at the origin of replication
•Helicase unwinds helix
•SSBPs prevent closure
•DNA gyrase reduces tension
•Core polymerase binds template
•DNA synthesis
•Not shown: pol I, ligase
Initiation of the replication of the bacterial chromosome
(called ORI)
Initiation of the replication of the Bacterial Chromosome
Initiation site is called oriC
BIDIRECTIONAL REPLICATION
Origin
5’
3’
5’
5’
3’
oriC
3’
RNA polymerase (RNAP) is specifically involved in starting DNA synthesis
at oriC.
ori
ter
RNAP initiates RNA synthesis within oriC. This opens up the double helix
at oriC. RNAP is sensitive to rifampicin.
All the primers for the both the leading strand and the Okazaki
fragments, needed later, are initiated by another RNA polymerase.
It is called RNA primase - it is resistant to rifampicin.
3’
5’
Initiation of the replication of the Bacterial Chromosome
Initiation site is called oriC
oriC
5’
3’
5’
3’
RNA polymerase starts to make RNA from two points
one on each strand, going in opposite directions from inside
oriC.
Initiation of the replication of the Bacterial Chromosome
Initiation and Termination of the replication of the
Bacterial Chromosome
BIDIRECTIONAL REPLICATION
oriC
Origin
3’
5’
leading
DNA
RNA
RNA
3’
DNA
leading
3’
5’
5’
3’
5’
1! After oriC is opened by RNAP, RNA primase starts to make
RNA from two points one on each strand, going in opposite directions
from inside oriC.
2 These are extended by DNA pol III as leading strands.
3 Okazaki fragments are made on the opposite strands.
ter
Replication Termination of the
Bacterial Chromosome
Procaryotic (Bacterial)
Cairns
Chromosome Replication
•! Termination: meeting of two replication forks
and the completion of daughter
chromosomes
ori
•! Region 180o from ori contains replication fork
traps:
ori
ter
Bidirectional Replication Produces
a Theta Intermediate
ori
Replication
Forks
Chromosome
Ter sites
Replication Termination of the Bacterial Chromosome
One set of Ter sites arrest DNA forks progressing
in the clockwise direction, a second set arrests forks
in the counterclockwise direction
Chromosome
TerB
TerA
Summary
•! Some of the DNA replication proteins:
DNA PolIII
DNA PolI
DNA Ligase
Primase (DnaG)
Helicase (DnaB)
SSB
DNA gyrase (topoisomerase)
•! Replication termination
Replication fork traps opposite oriC
Ter sites