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