Post on 19-Dec-2015
transcript
Replication
Which is the most necessary process for life?
• Is it translation ?
• Is it transcription ?
• Is it replication ?
DNA
RNA
Proteins
information flow
Information carryer replication
Energy
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
ChemistryP
P
P
P
P
P
P
P
CH2
CH2
CH2
OH
OH
O
O
OBase
Base
Base
CH2
CH2
CH2
OH
O
O
OBase
Base
Base
5' end of strand
3' end of strand3'
5'
3'
H20+
Synthesis reaction
OHO OHO
OHO OHO
OHO
OHO
OHO
OHO
OH
P PO O
OHOH
OHOH+
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
5’- GTCACC-3’ 5’- GTCACCG-3’ 3’-TTCAGTGGCAA-5’ 3’-TTCAGTGGCAA-5’
NEVER 3’-5’ polymerase activity!
+G
5’-3’ polymerase activity is present in all DNA and RNA polymerases
Enzymatic activities of polymerases
• 3’-5’ exonuclease (editing) activity
5’-AAGTCAC -3’ 5’-AAGTCAC-3’ 3’-TTCAGTGGCAA-5’ 3’-TTCAGTGGCAA-5’
A-A
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
5’-AA CACC-3’ 5’-AA CC-3’ 3’-TTCAGTGGCAA-5’ 3’-TTCAGTGGCAA-5’
A -ACA
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
Topoisomerase nicks DNA to relieve tension from unwinding
2
3
1
4
56
7
Pol III synthesises leading strand
Helicase opens helix
Primase synthesizes RNA primer
Pol III elongates primer; produces Okazaki fragment
Pol I replaces RNA primer with DNA
DNA ligase links two Okazaki fragments to form continuous strand
DNA REPLICATION (E.coli)
SSB protein prevents ssDNA from base-pairing
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 Function
2x 2x 2x 2x 2x ’
5’ to 3’ polymerizing activity3’ to 5’ exonuclease activity and assembly (scaffold)Assembly of holoenzyme on DNASliding clamp = processivity factorClamp-loading (“”) complex complex complex complex, binds to SSB complex
CoreEnzymedimer
Ho
loen
zym
e
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
Why a dimer?
Pol III =++
DNA looping during replication
How about eukaryotes?
• General mechanism of replication similar to prokaryotes with some minor differences
Topoisomerase nicks DNA to relieve tension from unwinding
2
3
1
5
56
7
Pol synthesises leading strand
Helicase opens helix
Primase synthesizes RNA primer
Pol replaces Pol ; produces Okazaki fragment
RNase H excises RNA primer
DNA ligase links two Okazaki fragments to form continuous strand
DNA REPLICATION (Eukaryotes)
4
Pol extends the RNA primer a little bit
RPA protein prevents ssDNA from base-pairing
Main differences among eukaryotic and prokaryotic
replication forks• In eukaryotes RNA primer is first extended by Pol
, then by Pol . In prokaryotes extension is done solely by Pol III
• In eukaryotes, RNA primer is excised by RNase H and then gap filled by Pol . 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)
Eukaryotic DNA polymerasesGreek Human Yeast Function
POLA POL1 Extension of RNA primer POLB - Base excision repair POLG MIP1 Mitochondrial replication POLD1 POL3 Main polymerase, like pol III in E.coli POLE POL2 Similar to , but not well understood POLZ REV3 Damage bypass POLH RAD30 Damage bypass POLQ - Interstrand cross-link repair POLI - Damage bypass POLK - Damage bypass POLL POL4 Joining dsDNA breakages POLM - Joining dsDNA breakages REV1 REV1 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
1 l culture = 4.1010 cells --> 400 000 km DNA synthesized (Earth-Moon distance)
Yeast 14 Mbp(1 cm)
3 kb/min 20 min 330 Repl. would last 80hr if only 1 ori
2.1013 km DNA synthesized (2 light-years) during life time (1016 cell divisions)
Human 3 Gbp(2 m)
3 kb/min 7 h >10 000 ? Repl. would last 1 year if only 1 ori
Genome Fork speed Repl. time Origins Comment
E. coli 4.6 Mbp 30 kb/min 40 min 1
Rate of DNA synthesis and the need for multiple origins
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 contain internal RNA, wich acts as a template
After one round of nucleotide addition, telomerase translocates to the next ttttgggg repeat
Telomerase in action
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
R-NH2 R=O[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
deoxyadenosineN-6-methyldeoxyadenosine
....... .......
......
.
......
.
Dam methylation
CACGATCCATT
GTGCTAGGTAA
CACGATC ATT
GTGCTAG TAA
CACGATCCATT
GTGCTAGGTAA
Replication
CH3
CH3
CH3
CH3
C
T
Error
Correct
Replication machinery recognizes the methylated strand and corrects the other strand. This is valid for prokaryotes, mechanism for eukaryotes has not been established yet
Dam methylation in E.coli : A’s in GATC sequences get methylated
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?
• DNA replicates only during S phase and only once
• This implies some sort of switch...
• Cyclins take care of thatGo
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, 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.
ORC
origin
DNA
Origin Recognition Complex
CDC6 and Cdt1 proteins are expressed only during S-phase and they bind to ORC
ORC
origin
DNA
CDC6 and Cdt1 proteins
CDC6
Cdt1
CDC6 and Cdt1 bring the MCM2-7 helicase to the origin The whole complex still needs activation
ORC
origin
DNA
MCM2-7 helicase
MCM2-7
CDC6
Cdt1
Now the complex can activate replication
ORC
origin
DNA
Phosphorylation of initiation complex
MCM2-7P
P
Cycline dependent kinases phosphorylate the complexCdt1
CDC6P P
P
MCM2-7 moves along the DNA and opens the double helix. Other replication proteins can come into action now
ORC
origin
DNA
Initiation
MCM2-7
P
P
To prevent further initiation rounds, Geminin protein binds to CDC6 and CDT1, blocking binding of another MCM2-7
Cdt1
CDC6P P
Geminin
P
ORC
CDC6
Cdt1P
P
P
P
GemininORC
ORC
CDC6
Cdt1
ORC
CDC6
Cdt1P
P
P
P
MCM2-7
MCM2-7
ORC
CDC6
Cdt1Geminin
The Switch.
G1
early S-phase S-phase
S-phaselate S-phase/mitosis
mitosis
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 forksand the completion of daughter chromosomes
Region 180o from ori contains replication forktraps:
ori
Ter sites
Chromosome
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:
TerATerB
Chromosome
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
TusDNA
Ter
Replication forkarrested in polar
manner
Tus may inhibit replication fork progressionby 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