Transcript Repliction - Uppsala University

Replication
Which is the most necessary
process for life?
• Is it translation ?
• Is it transcription ?
• Is it replication ?
Energy
DNA
RNA
Proteins
information flow
Information carryer replication
Outline
• Overview
• Replication fork and involved enzymes
• Differences among eukaryotes and
prokaryotes
• DNA repair
• Replication initiation
• Replication termination
What happens upon
replication?
1. Double-stranded DNA
unwinds
2. Two new strands are
formed by pairing
complementary bases
with the old strands
Chemistry
O
P
OH
CH2
O
P
O
P
O
OH
O
OH
CH2
Base
O
CH2
5' end of strand
Base
O
P
OH
CH2
Base
Base
O
OH
O
P
O
OH
3'
P
OH
O
OH
P
O
O
Synthesis reaction
OH
5' CH2
3'
OH
O
P
OH
CH2
+
O
OH
OH
P
P
H 20
+
O
OH OH
Base
Base
OH
3' end of strand
What do you need for replication ?
• 1) template - dsDNA
• 2) Origin - some place in dsDNA, which is
recognized by replication machinery
• 3) polymerse & other replicating enzymes
• 4) nucleotides
Enzymatic activities of
polymerases
• 5’-3’ polymerase activity
+G
5’- GTCACC-3’
3’-TTCAGTGGCAA-5’
5’- GTCACCG-3’
3’-TTCAGTGGCAA-5’
NEVER 3’-5’ polymerase activity!
5’-3’ polymerase activity is present in all DNA and RNA
polymerases
Enzymatic activities of
polymerases
• 3’-5’ exonuclease (editing) activity
5’-AAGTCAC A-3’
3’-TTCAGTGGCAA-5’
-A
5’-AAGTCAC-3’
3’-TTCAGTGGCAA-5’
Normally, only one mismatched nucleotide is removed
3’-5’ exonuclease activity is present in most (but not all)
DNA and RNA polymerases
Enzymatic activities of
polymerases
• 5’-3’ exonuclease activity
-ACA
A
5’-AA CACC-3’
5’-AA
CC-3’
3’-TTCAGTGGCAA-5’
3’-TTCAGTGGCAA-5’
5’-3’ exo activity requires a free 5’-end or a nick in dsDNA
5’-3’ exo activity can be combined with 5’-3’ polymerase
activity. This results in a replacement of a part of strand.
5’-3’ exo activity is present only in some DNA polymerases,
notably bacterial DNA polymerase I
Replication enzymes (summary)
•
•
•
•
•
•
Polymerase III (E.coli) – adds nucleotides
Helicase – unwinds the DNA
Topoisomerase – releases tension on ds DNA
SSB – binds to ssDNA
Primase – makes RNA primer
Polymerase I (E.coli)– replaces RNA primers with
DNA
• Ligase – joins Okazaki fragments
DNA REPLICATION (E.coli)
4
2
Pol III synthesises leading strand
1 Helicase opens helix
3 Primase synthesizes RNA primer
Topoisomerase
nicks DNA to
relieve tension
from unwinding
SSB protein
prevents ssDNA
from basepairing
5
Pol I replaces RNA
primer with DNA
6
7
Pol III elongates primer;
produces Okazaki fragment
DNA ligase links
two Okazaki
fragments to form
continuous strand
DNA polymerases in E.coli
•
•
•
•
•
DNA pol I – excises RNA primer and fills the gap
DNA pol II – DNA repair
DNA pol III – main replicating enzyme
Recently discovered:
DNA pol IV – increase mutation rate upon
starvation and stress conditions (“Mutate or die!”)
• DNA pol V – “SOS” polymerase, active upon
DNA- damaging conditions. Can bypass damaged
DNA effectively at a cost of higher mutation rate
(“Replicate or die”).
The subunits of E. coli DNA polymerase III
Subunit
Holoenzyme
Core
Enzyme
dimer
2x a
2x e
2x q
2x t
2x b
g
d
d’
c
y
Function
5’ to 3’ polymerizing activity
3’ to 5’ exonuclease activity
a and e assembly (scaffold)
Assembly of holoenzyme on DNA
Sliding clamp = processivity factor
Clamp-loading (“g”) complex
g complex
g complex
g complex, binds to SSB
g complex
Sliding clamp around the DNA
Clamp ensures processivity of
nucleotide addition
Clamp is loaded only once on
the leading strand
Clamp is re-loaded on the
lagging strand upon synthesis
of new Okazaki fragment
Structure of clamp
•
•
•
•
pseudo-6-fold symmetry
prokaryotes – dimer
eukaryotes – trimer
Domains within the monomer have very similar
structure but no detectable sequence similarity
Subunits of pol III in E.coli
Pol III =a+e+q
Why a dimer?
DNA looping during replication
How about eukaryotes?
• General mechanism of replication similar to
prokaryotes with some minor differences
DNA REPLICATION (Eukaryotes)
RPA protein
prevents ssDNA
from base-pairing
5
2
Topoisomerase
nicks DNA to
relieve tension
from unwinding
Pol d synthesises leading strand
1 Helicase opens helix
3Primase synthesizes RNA primer
4
Pol a extends the
RNA primer a little
bit
Pol d replaces Pol a;
produces Okazaki fragment
5
RNase H excises
6 RNA primer
7
DNA ligase links
two Okazaki
fragments to form
continuous strand
Main differences among eukaryotic
and prokaryotic replication forks
• In eukaryotes RNA primer is first extended by Pol
a, then by Pol d. In prokaryotes extension is done
solely by Pol III
• In eukaryotes, RNA primer is excised by RNase H
and then gap filled by Pol d. In prokaryotes Pol I
is able to both excise RNA and fill in DNA
• Okazaki fragments in eukaryotes are about 200 nt
long, while in bacteria 2000 nt (yes, not the other
way around)
Greek
a
b
g
d
e
z
h
q
i
k
l
m
-
Eukaryotic DNA polymerases
Human
POLA
POLB
POLG
POLD1
POLE
POLZ
POLH
POLQ
POLI
POLK
POLL
POLM
REV1
Yeast
POL1
MIP1
POL3
POL2
REV3
RAD30
POL4
REV1
Function
Extension of RNA primer
Base excision repair
Mitochondrial replication
Main polymerase, like pol III in E.coli
Similar to d, but not well understood
Damage bypass
Damage bypass
Interstrand cross-link repair
Damage bypass
Damage bypass
Joining dsDNA breakages
Joining dsDNA breakages
Damage bypass
Reasons for differences in replication
among prokaryotes and eukaryotes
• 1. Eukaryotic chromosomes are typically
much longer than prokaryotic
• 2. Eukaryotic chromosomes are linear, not
circular
Multiple origins in chromosomes
Bacteria
Eukaryotes
Rate of DNA synthesis and the need for
multiple origins
Genome Fork speed
E. coli
4.6 Mbp
30 kb/min
Repl. time Origins
40 min
Comment
1
Repl. would last 80hr
14 Mbp
3 kb/min
20 min
330
if only 1 ori
(1 cm)
1 l culture = 4.1010 cells --> 400 000 km DNA synthesized (Earth-Moon distance)
Yeast
3 Gbp
3 kb/min
7h
>10 000 ? Repl. would last
1 year if only 1 ori
(2 m)
2.1013 km DNA synthesized (2 light-years) during life time (1016 cell divisions)
Human
Linear DNA needs special treatment:
Telomeres and telomerases
• Telomeres: short, repetitive sequences in the
ends of eukaryotic chromosomes
• Telomerase: polymerase, making those
sequences
• What are they good for?
Telomerase in action
Telomerase contain internal RNA, wich acts as a template
After one round of nucleotide addition, telomerase translocates to
the next ttttgggg repeat
T-loops
TRF1 and 2 – telomere
binding proteins
Formation of T-loops
controls the lenght of
telomeres
Is telomerase always active?
• Active in children and germ cells of adults
• Inactive in somatic cells of adults
• So, chromosomes actually get shorter – this is why
we get old and die...
• For the same reason, cultivated primary animal
cells do not divide infinitely
• Activation of telomerase in adult mice increase
their life span
• Telomerase is active in most tumours
DNA damage
• 1. Base damage: deamination, depurination,
alkylation...
• 2. Thymine dimerisation
• DNA damage can lead to:
- 1. prevention of base pairing
- 2. incorrect base paring
• Those types of DNA damage are NOT caused by
DNA polymerase errors
Deamination
[O]
R-NH2  R=O
Thymine dimers
Produced by UV light
Results in no base-pairing with the complementary
strand
Repair of
damaged bases
Repair of G-T and G-U base pairs
• The most usual mutation is
deamination of cytosine or
methylcytosine
• As a result, uracil or thymine is
produced, which both base-pair to
adenine
• Special repair mechanism has been
developed for this mutation
Excision of thymine dimers
How do those repair enzymes
know, which strand to repair?
• Upon introduction of mutation in one
strand, a mismatch is produced:
• The template strand has to be distinquished
from the newly made strand
• In prokaryotes template strand has been
previously labelled by methylation
.......
.......
Dam methylation
.......
deoxyadenosine
.......
N-6-methyldeoxyadenosine
Dam methylation
Dam methylation in E.coli :
A’s in GATC sequences get
methylated
CH3
CACGATCCATT
Replication
GTGCTAGGTAA
CH3
CH3
C
CACGATC ATT
GTGCTAG TAA
T
CACGATCCATT
GTGCTAGGTAA
CH3
Replication machinery recognizes the methylated strand and corrects
the other strand. This is valid for prokaryotes, mechanism for
eukaryotes has not been established yet
Repair of dsDNA breaks
• Under certain mutagenic conditions, break
of dsDNA can occur
• If this happens during late S or G2 phase,
the sister chromatid is around which can be
used as a reference
• Otherwise – error prone ligation is a option
(can be dangerous!)
DNA damage bypass
• Necessary, if a replication fork reaches
damaged region of DNA
• Two main types of bypass exist:
• 1. Bypass by recombination
• 2. Translesion
Bypass by recombination
• Damage (blue circle)
hopefully occurs only in
one parental strand
• Newly made DNA strand
temporarily base-pairs with
the other newly made
strand
Translesion
• Damage (lesion) bypass without information of
other parental strand
• Can be mutagenic or unmutagenic
• In humans, polymerase eta is responsible for
translession past thymine dimers
• Individuals, lacking eta pol, use alternative,
thymine dimer translesion pathway by pol zeta
• zeta pathway is more mutagenic than eta
• As a result, risk of cancer development under UV
exposure is significantly increased
Error rates during replication
• DNA pol without proofreading: 1:105
• DNA pol with proofreading: 1:107
• Most errors will be corrected by repair
enzymes. This leaves error rate of 1:1010
• Since human genome is 3.2x109 base pairs
long, about one mutation is made upon each
genome replication
Question
• Errors in replication can lead to cancer,
genetic diseases, etc
• Why Mother Nature has not eliminated
DNA replication errors completely ?
• Or at least, why the error rate has not been
decreased still more ?
When to replicate?
Go
• DNA replicates only
during S phase and only
once
• This implies some sort
of switch...
• Cyclins take care of that
What are those cyclins anyway?
• Cyclins are proteins, which give a signal
that it is time to proceed to the next cell
cycle phase
• Cyclins bind to and activate cyclin
dependent kinases (CDKs)
• CDKs phosphorylate and thereby activate
various regulatory proteins
Origin Recognition Complex
ORC
DNA
origin
Origin Recognition Complex (ORC, six subunits) binds
specifically to origin DNA sites on the chromosome.
ORC is bound to origin DNA regardless of whether
replication is occurring or not.
CDC6 and Cdt1 proteins
CDC6
ORC
Cdt1
DNA
origin
CDC6 and Cdt1 proteins are expressed only during Sphase and they bind to ORC
MCM2-7 helicase
CDC6
ORC
Cdt1
MCM2-7
DNA
origin
CDC6 and Cdt1 bring the MCM2-7 helicase to the origin
The whole complex still needs activation
Phosphorylation of initiation complex
ORC
P
P
CDC6
Cdt1
P
P
MCM2-7
DNA
origin
Now the complex can activate replication
P
Cycline dependent
kinases
phosphorylate the
complex
Initiation
ORC
P
P
P
CDC6
Geminin
Cdt1
P
MCM2-7
DNA
origin
P
MCM2-7 moves along the DNA and opens the double
helix. Other replication proteins can come into action now
To prevent further initiation rounds, Geminin protein binds to
CDC6 and CDT1, blocking binding of another MCM2-7
The Switch.
P
CDC6
CDC6
Cdt1
ORC
P
Cdt1
ORC P
MCM2-7
P
early S-phase
ORC
G1
mitosis
CDC6
Geminin
Cdt1
ORC
late S-phase/
mitosis
S-phase
P
CDC6
Geminin
P
Cdt1
ORC P
MCM2-7
P
S-phase
Replication termination
• Not well understood, particularly in
eukaryotes (where it maybe do not exist...)
• In prokaryotes, replication termination
sequences are found opposite the origin
Replication Termination of the
Bacterial Chromosome
 Termination: meeting of two replication forks
and the completion of daughter chromosomes
 Region 180o from ori contains replication fork
traps:
ori
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
As a result, replication
forks bypass each other a
bit and thus make
slightly longer sequence
than necessary
Replication Termination of the
Bacterial Chromosome
 Ter sites are binding sites for the Tus protein
Tus
DNA
Replication fork
arrested in polar
manner
Ter
 Tus may inhibit replication fork progression
by directly contacting DnaB helicase,
inhibiting DNA unwinding
After termination
• The strands must be joined together
somehow
• How? I don’t know...
Decatenation (prokaryotes only)
After replication of circular DNA, the two daughter DNA circles
are interlocked
Topisomerase IV opens one chromosome and re-ligates after
chomosome separation