DNA Replication - PW

Cen

tral

Dog

ma

Nucleic acid

Lipid

Protein

Nucleic acid

Nucleic acid

Nucleic acid

+ proteinNucleic acid

DNA Replication

•Molecular Biology Course

Eukaryotic DNA replicationEukaryotic DNA replication

•Molecular Biology Course

DNA Replication: An OverviewDNA Replication: An OverviewReplicons, semi-conservative, semi-discontinous, RNA priming

Bacterial DNA replicationBacterial DNA replicationExperimental system, initiation, unwinding, elongation, termination & segregation

Experimental system, cell cycle, initiation, replication forks, nuclear matrix, telomere repl.

•DNA replication

DNA Replication: An Overview

DNA Replication: An Overview

1.Replicons 2.semi-conservative

mechanism3.semi-discontinous

replication4.RNA priming

Replicon is any piece of DNA which replicates as a single unit. It contains an origin and sometimes a terminus

Origin is the DNA sequence where a replicon initiates its replication.Terminus is the DNA sequence where a replicon usually stops its replication

•DNA replication

Replicons

rep gene

Ope

rato

r site

Itero

ns

DnaA b

oxes

AT-rich

RepliconOrigin

Prokaryotic genome: a single circular DNA = a single replicon

Eukaryotic genome: multiple linear chromosomes & multiple replicons on each chromosome

•DNA replication

Origin

5’3’

3’5’

UNIDIRECTIONAL REPLICATION

Origin

5’3’

3’5’

BIDIRECTIONAL REPLICATION

Replication can be Uni- or Bidirectional

Bidirectional replication of a circular bacterial replicon

• All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circlular and comprise single replicons. • There is a single termination site roughly 180o opposite the unique origin.

•DNA replication

Linear viral DNA molecules usually have a single origin.

In all the cases, the origin is a complex region where the initiation of DNA replication and the control of the growth cycle of the organism are regulated and co-ordinated.

•DNA replication

The long, linear DNA molecules of eukaryotic chromosomes consist of mutiple regions, each with its own origin.

A typical mammalian cell has 50000-100000 replicons with a size range of 40-200 kb. When replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required

Multiple eukaryotic replicons and replication bubbles

•DNA replication

Multiple origins of replication in Eukaryotes

+

Are all origins created equal?

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 S 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 ? S would last 1 year if 1 ori

Genome Fork speed S phase Origins Comment

E. coli 4.6 Mbp 30 kb/min 40 min 1 S longer than doubling time

Rate of DNA synthesis and the need for multiple origins

Procaryotic (Bacterial) and Eucaryotic Chromosome Replication

ori

ter

BACTERIAL CHROMOSOME

EUCARYOTIC CHROMOSOME

ori ori ori

•DNA replication

replication bubbles replication fork

•DNA replication

Replication is Semi-conservative

• In the late 1950s, three different mechanisms were proposed for the replication of DNA– Conservative model

• Both parental strands stay together after DNA replication

– Semiconservative model• The double-stranded DNA contains one parental and one

daughter strand following replication

– Dispersive model• Parental and daughter DNA are interspersed in both strands

following replication

Proposed Models of DNA ReplicationProposed Models of DNA Replication

Alternative models of DNA replication :

Equilibrium density gradient centrifugation

15N14N

DNA

• Matthew Meselson and Franklin Stahl experiment in 1958– Grow E. coli in the presence of 15N (a heavy isotope of

Nitrogen) for many generations• Cells get heavy-labeled DNA

– Switch to medium containing only 14N (a light isotope of Nitrogen)

– Collect sample of cells after various times– Analyze the density of the DNA by centrifugation using

a CsCl gradient

1958: Matthew Meselson & Frank Stahl’s ExperimentSemiconservative model of DNA replication

Interpreting the Data

After one generation, DNA is “half-

heavy”

After ~ two generations, DNA is of two types: “light” and

“half-heavy”

This is consistent with only the semi-conservative model

•DNA replication

Replication is Semi-discontinuous

Semi-discontinuous replication

Ligation

•DNA replication

Okazaki fragments

DNA Synthesis is Semidiscontinous

5’3’5’3’

3’5’

Lagging strand synthesis

Leading strand synthesis

Okazaki Fragment Synthesis

Leading strand synthesis

Primase synthesizes RNA primer

DNA PolIII extendsprimer into Okazakifragment

Okazaki Fragment Synthesis (cont.)

New Okazaki fragment

DNA PolI displaces RNA primer with DNA

Gap sealed by DNA ligase

DNA replication is continuous on the leading strand and semidiscontinuous on the lagging strand:

Unwinding of any single DNA replication fork proceeds in one direction.

The two DNA strands are of opposite polarity, and DNA polymerases only synthesize DNA 5’ to 3’.

Solution: DNA is made in opposite directions on each template.

•Leading strand synthesized 5’ to 3’ in the direction of the replication fork movement.continuous

requires a single RNA primer

•Lagging strand synthesized 5’ to 3’ in the opposite direction.semidiscontinuous (i.e., not continuous)requires many RNA primers

3

Polymerase III

5’ 3’

Leading strand

base pairs

5’

5’

3’

3’

Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:

Helicase +

Initiator Proteins

ATP

SSB Proteins

RNA Primer

primase

2Polymerase III

Lagging strand

Okazaki Fragments

1

RNA primer replaced by polymerase I

& gap is sealed by ligase

DNA ligase seals the gaps between Okazaki fragments with aphosphodiester bond

Discovery of Okazaki fragmentsEvidence for semi-discontinuous

replication

[3H] thymidine pulse-chase labeling experiment1. Grow E. coli2. Add [3H] thymidine in the medium for a few

second spin down and break the cell to stop labeling analyze found a large fraction of nascent DNA (1000-2000 nt) = Okazaki fragments

3. Grow the cell in regular medium then analyze the small fragments join into high molecular weight DNA = Ligation of the Okazaki fragments

•DNA replication

DNA Synthesis Occurs in the 5’3’ Direction

P P P P P PP PP

PP PP5’

3’ 5’

OH 3’

OH 3’

P P P P P PP PP

PP PP5’

3’ 5’

OH3’5’PPP

P P P P P PP PP

5’

3’ 5’

OH 3’PP PP P

PP

Incoming nuceolotidetriphosphate

Nucleotide monophosphateadded to chain with release

of diphosphate

•DNA replication

RNA priming

The first few nucleotides at the 5’-end of Okazaki fragments are ribonucleotides. Hence, DNA synthesis is primed by RNA that is then removed before fragments are joined. Crucial for high fidelity of replication

Fidelity Of DNA Replication

DNA replication is extremely accurate - one error for every 109 bpreplicated• But some non-Watson/Crick basepairs such as this GT basepair are only about 100-fold less stable than a normal Watson/Crick basepair.• This suggests that the error rate of DNA replication should be one in a hundred instead of one in a billion• So how do we explain the low error rate?

Low error frequency accounted for by redundant safeguards1. Binding pocket of DNA polymerase clamps tightly around the base before catalysis occurs. Wobble pairs don’t fit and so catalysis can’t occur.2. DNA polymerases have “editing exonuclease activities” that allow them to erase mistakes and try again3. Cells contain mismatch repair systems that come along after DNA polymerase to clean up any remaining errors.Each of the above safeguards improves accuracy by about 2-3 orders of magnitude, thus explaining the overall 10-9 error frequency.

•DNA replication

Enzymes/Proteins Involved

The Major DNA Polymerases

BACTERIAL

Enzyme Primary function

DNA Pol I (PolA) Major DNA repair enzymeDNA Pol II DNA repairDNA Pol III De novo synthesis of new DNA

_______________________________________________

MAMMALIAN

Enzyme Primary functionLocation

DNA Pol I () Strand synthesis initiationNucleus

DNA Pol II () DNA repairNucleusDNA Pol III () Strand extensionNucleusDNA Pol DNA repair

NucleusDNA Pol De novo synthesis of new DNA

Mitochon.

5’3’ synthesis

Nucleotide misincorporation

Polymerase reversal and 3’5’ exonuclease activity

Continued 5’3’ synthesis

DNA polymerase

Incorrect nucleotide

Some DNApolymerases have 3’5’ exonuclease activity

Proteins at the Replication Fork

Parental DNA

Leading strand

Lagging strand (Okazaki fragment)

5’3’

3’5’

3’5’

DNA polIII

Single-stranded binding (SSB) protein

Primase

DNA helicase

RNA primer

+ DNA PolI+ Ligase

DNA Polymerase III

5’3’5’3’

3’5’

Primary replicative DNA polymerase in E. coli

Catalyzes DNA chain elongation by the formation of phosphodiester bonds

Incoming dNTP is positioned for chain incorporation by H-bonding with template nucleotide

Can only extend chains from 3’OH termini, cannot initiate synthesis of new chains

Catalyzes leading and lagging strand syntheses

OH 3’

P P P P P PP PP

PP PP5’

3’ 5’

OH3’5’PPP

DNA Pol III Catalyzes Phosphodiester Bond Formation in a DNA Chain

Some Important Features of DNA PolIII Dimeric: one monomer associated with leading strand, other with lagging strand

130 kD monomer (also known as subunit)

Functions as part of DNA PolII holoenzyme complex which contains 10 subunits

Subunits reaction rate and processivity:reaction rate ~1000nts/secprocessitivity 5 x 105

Processivity is due to ‘sliding clamp’ of subunit:

5’ 3’

DNA Polymerase I

103 kD

Catalyzes DNA chain elongation by the formation of phosphodiester bonds

Incoming dNTP is positioned for chain incorporation by H-bonding with template nucleotide

Can only extend chains from 3’OH termini, cannot initiate synthesis of new chains

Catalyzes lagging strand synthesis

A proofreading activity which results in the removal of improperly-paired nucleotide

1. 5’3’ DNA chain synthesis:

2. 3’ exonuclease activity:

DNA Polymerase I (continued)

Excision of RNA primer from Okazaki fragment during lagging strand synthesis

5’ 3’

Okazaki fragment RNA primer

Nick

3. 5’ exonuclease activity:

Two problems posed by the properties of the known DNA polymerases

1. The directionality problem. How can DNA polymerase replicate both strands behind each replication fork, when all polymerases operate in the 5’ to 3’ direction? Solution - semidiscontinuous DNA synthesis

2. The priming problem. Since all DNA polymerases require a primer (usually of at least 10 nucleotides in length), where do the primers come from? Solution - primers are made of RNA

DNA Ligase

75 kD

Following complete replacement of RNA primer by DNA in lagging strand synthesis, a nick with 3’OH and a 5’phosphate end is generated

DNA ligase catalyzes phosphodiester bondformation on nick:

5’5’3’3’

5’ 3’HOP

5’3’ 5’

3’P

Primase (DnaG) 60 kD

Intiates Okazaki fragment synthesis from a single-stranded DNA template:

After addition of 10-12 ribonucleotides, primase is displaced by DNA PolIII which synthesizes DNA from the 3’OH group on RNA primer

Complexed with helicase in lagging strand

Primase

RNA primer

Helicase (DnaB)

Hexameric (6 x 50 kD)

Catalyzes unwinding of DNA duplex thereby exposing single stranded DNA

Different helicases with different polarities on two strands in duplex

ATP hydrolysis provides energy for unwinding

DNA Helicase Separates Strands

Helicase Binds to DNA Polymerase III

Single Stranded Binding Protein (SSB)

Tetrameric (4 x 19 kD)

Binds to single-stranded DNA and prevents duplex reannealing

>1000-fold affinity for single-stranded DNA compared to double-stranded DNA

Lowers DNA melting temperature, i.e., promotes DNA denaturation

Binding is cooperative resulting in coating of the single-stranded DNA:

SSBssDNA

Proteins at the Replication Fork

Parental DNA

Leading strand


5’3’

3’5’

3’5’

DNA polIII

Single-stranded binding (SSB) protein

Primase

DNA helicase

RNA primer

+ DNA PolI+ Ligase

•DNA replication

Bacterial DNA replication

Bacterial DNA replication

1. Experimental system 2. initiation, 3. unwinding, 4. elongation, 5. termination & segregation

• DNA synthesis begins at a site termed the origin of replicationorigin of replication

• Each bacterial chromosome has only one

• Synthesis of DNA proceeds bidirectionallybidirectionally around the bacterial chromosome– eventually meeting at the opposite side of the

bacterial chromosome• Where replication ends

BACTERIAL REPLICATION BACTERIAL REPLICATION

1955: Arthur Kornberg

Worked with E. coli. Discovered the mechanisms of DNA synthesis.

Four components are required:

1. dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside 5’-triphosphates)(sugar-base + 3 phosphates)

2. DNA template

3. DNA polymerase I (formerly the Kornberg enzyme)(DNA polymerase II & III discovered soon after)

4. Mg 2+ (optimizes DNA polymerase activity)

Three main features of the DNA synthesis reaction:

1. DNA polymerase I catalyzes formation of phosphodiester bond between 3’-OH of the deoxyribose (on the last nucleotide) and the 5’-phosphate of the dNTP.

• Energy for this reaction is derived from the release of two of the three phosphates.

2. DNA polymerase I “finds” the correct complementary dNTP at each step in the lengthening process.

• rate ≤ 800 dNTPs/second• low error rate

3. Direction of synthesis is 5’ to 3’

Not all polymerases are the same

Polymerase Polymerization (5’-3’) Exonuclease (3’-5’) Exonuclease (5’-3’) #Copies

I Yes Yes Yes 400

II Yes Yes No ?

III Yes Yes No 10-20

•3’ to 5’ exonuclease activity = ability to remove nucleotides from the 3’ end of the chain

•Important proofreading ability

•Without proofreading error rate (mutation rate) is 1 x 10-6

•With proofreading error rate is 1 x 10-9 (1000-fold decrease)

•5’ to 3’ exonuclease activity functions in DNA replication & repair.

Replication of circular DNA inE. coli :

1. Two replication forks result in a theta-like () structure.

2. As strands separate, positive supercoils form elsewhere in the molecule.

3. Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate.

Rolling circle model of DNA replication :

1. Common in several bacteriophages including .

2. Begins with a nick at the origin of replication.

3. 5’ end of the molecule is displaced and acts as primer for DNA synthesis.

4. Can result in a DNA molecule many multiples of the genome length (and make multiple copies quickly).

5. During viral assembly the DNA is cut into individual viral chromosomes.

In vitro experimental systems

1. Purified DNA: smaller and simpler bacteriophage and plasmid DNA molecules (X174, 5 Kb)

2. All the proteins and other factors for its complete replications

•DNA replication

In vitro system: Put DNA and protein together to ask for replication question

•DNA replication

Initiation

• The origin of replication in E. coli is termed oriC

–origin of Chromosomal replication

• Important DNA sequences in oriC

–AT-rich regionAT-rich region

–DnaA boxesDnaA boxes

1. oriC contains four 9 bp binding sites for the initiator protein DnaA. Synthesis of DnaA is coupled to growth rate so that initiation of replication is also coupled to growth rate.

2. DnaA forms a complex of 30-40 molecules, facilitating melting of three 13 bp AT-rich repeat sequence for DnaB binding.

3. DnaB is a helicase that use the energy of DNA hydrolysis to further melt the double-stranded DNA .

4. Ssb (single-stranded binding protein) coats the unwinded DNA.

5. DNA primase load to synthesizes a short RNA primer for synthesis of the leading strand.

6. Primosome: DnaB helicase and DNA primase

Origin of replication (e.g., the prokaryote example):

Begins with double-helix denaturing into single-strands thus exposing the bases.

Exposes a replication bubble from which replication proceeds in both directions.

~245 bp in E. coli

Initiation

Initiation of replication, major elements:

Segments of single-stranded DNA are called template strands.

Gyrase (a type of topoisomerase) relaxes the supercoiling in DNA generated ahead of each replication fork.

Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate).(Hydrolysis of ATP causes a shape change in DNA helicase)

DNA primase next binds to helicase producing a complex called a primosome (primase is required for synthesis),

Initiation of replication, major elements:

Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase III adds nucleotides.

Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at the RNA primer.

The RNA primer is removed and replaced with DNA by polymerase I, and the gap is sealed with DNA ligase.

Single-stranded DNA-binding (SSB) proteins (>200) stabilize the single-stranded template DNA during the process.

Fig. 11.9a(TE Art)

Able to covalently linktogether

Unable to covalently link the 2 individualnucleotides together

Primer

5’

5’

5’

5’

5’

5’

3’

3’

3’

3’

3’

DNA Polymerase Cannot Initiate new Strands

Synthesis and replacement of RNA primers during DNA replicationSynthesis and replacement of RNA primers during DNA replication

Initiation of Replication at oriCInitiation of Replication at oriC

• DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences

– causes the region to wrap around the DnaA proteins and separates the AT-rich region

Uses energy from ATP to unwind the duplex DNA

SSB

SSB SSB

SSB

Re-initiation of bacterial replication at new origins before completion of the first round of replication

Positive supercoiling: caused by removal of helical turns at the replication fork.

Resolved by a type II topoisomerase called DNA gyrase

•DNA replication

Unwinding

Topoisomerase at the Replication Fork

•DNA replication

Elongation

“Three Dimensional” view of Replication Fork

Direction of fork movement

Direction of synthesis Of lagging strand

Direction of synthesis of leading strand

DNA polymerase III holoenzyme: 1. a dimer complex, one half synthesizing the leading

strand and the other lagging strand.2. Having two polymerases in a single complex ensures

that both strands are synthesized at the same rate3. Both polymerases contain an -subunit---polymerase

-subunit---3’5’ proofreading exonuclease -subunit---clamp the polymerase to DNA

other subunits are different.

Replisome: in vivo, DNA polymerase holoenzyme dimer, primosome (helicase) are physically associated in a large complex to synthesize DNA at a rate of 900 bp/sec.

Other two enzymes during Elongation

1. Removal of RNA primer, and gap filling with DNA pol I

2. Ligation of Okazaki fragments are linked by DNA ligase.

Components Of The E. Coli Replisome

1. Helicases - Unwind DNA at the replication fork in a reaction coupled to ATP Hydrolyis2. Single-stranded DNA binding proteins (SSB) - Bind and stabilize the DNA in a single stranded conformation after the melting by helicases3. Primosome - Synthesizes RNA primers for the lagging strand4. DNA Polymerase III - The replicase5. Type II Topoisomerase (Gyrase) - Relaxes postively supercoiled DNA that forms ahead of the replication fork. Decatenates the final product6. DNA Polymerase I - Replaces RNA primers with DNA by nicktranslation7. DNA Ligase - Joins the Okazaki fragments

Elongation: lagging strand replication

Polymerase III holoenzyme(DNA pol III)

DNA pol I (5’3’ exonulclease activity)

DNA pol I (5’3’ polymerase activity)

DNA ligase

Directionality of the DNA strands at a replication forkDirectionality of the DNA strands at a replication fork

Leading strand

Lagging strand

Fork movementFork movement

DNA elongation :

•DNA replication

Termination and Segregation

•Terminus: containing several terminator sites (ter) approximately 180o opposite oirC.•Tus protein: ter binding protein, an inhibitor of the DnaB helicase

Termination

ter

ori

ter

Replication Termination of the Bacterial Chromosome

Origin

5’3’

3’5’

BIDIRECTIONAL REPLICATION

Termination: meeting of two replication forks and the completion of daughter chromosomes

Region 180o from ori contains replication fork traps:

ori

Ter sites

Chromosome


TerATerB

One set of Ter sites arrest DNA forks progressing in the clockwise direction, a second set arrests forks in the counterclockwise direction:

Chromosome


Ter sites are binding sites for the Tus protein

Tus:35.8 kDDNA binding at TerMonomer

Tus

DNA

Ter

Replication forkarrested in polar

manner

Tus may inhibit replication fork progression by directly contacting DnaB helicase, inhibiting DNA unwinding


•Topoisomerase IV: a type II DNA topoisomerase, function to unlink the interlinked daughter genomes.

Segregation

Model of replication in E. coli

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Model for the events occurring around a single replication fork of the E. coli chromosome


Model for the events occurring around a single replication fork of the E. coli chromosome

Trombone Model of DNA Replication in E. coli I

Trombone Model of DNA Replication in E. coli II

Trombone Model of DNA Replication in E. coli III

Trombone Model of DNA Replication in E. coli IV

Trombone Model of DNA Replication in E. coli V

Concepts and terms to understand:

Why are gyrase and helicase required?

The difference between a template and a primer?

The difference between primase and polymerase?

What is a replication fork and how many are there?

Why are single-stranded binding (SSB) proteins required?

How does synthesis differ on leading strand and lagging strand?

Which is continuous and semi-discontinuous?

What are Okazaki fragments?

•DNA replication

Eukaryotic DNA replication

Eukaryotic DNA replication1. Experimental system2. cell cycle, 3. initiation, 4. replication forks, 5. nuclear matrix, 6. telomere replication.

DNA replication in eukaryotes:

Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges:

Major checkpoints in the system

1. Cells must be large enough, and the environment favorable.

2. Cell will not enter the mitotic phase unless all the DNA has replicated.

3. Chromosomes also must be attached to the mitotic spindle for mitosis to complete.

4. Checkpoints in the system include proteins call cyclins and enzymes called cyclin-dependent kinases (Cdks).

• Each eukaryotic chromosome is one linear DNA double helix

• Average ~108 base pairs long

• With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days.

• Solution ---> DNA replication initiates at many different sites simultaneously.

Rates are cell specific!

Eukaryotic enzymes:

Five DNA polymerases from mammals.

1. Polymerase (alpha): nuclear, DNA replication, no proofreading

2. Polymerase (beta): nuclear, DNA repair, no proofreading

3. Polymerase (gamma): mitochondria, DNA repl., proofreading

4. Polymerase (delta): nuclear, DNA replication, proofreading

5. Polymerase (epsilon): nuclear, DNA repair (?), proofreading

• Different polymerases for nucleus and mtDNA

• Some proofread; others do not.

• Some used for replication; others for repair.

• Pol δ - the eukaryotic replicase

• Pol α/primase - contains both primase and DNA polymeraseActivities

• PCNA - trimeric sliding clamp

• Replication Factor C (RFC) – the clamp loader

• MCMs - a heterohexameric helicase

• Replication Protein A (RPA) = SSB

• RNase H - nuclease that is specific for RNA in RNA/DNA hybrids - excises primers

In vitro experimental systems

1. Purified DNA : 2. All the proteins and other factors for its

complete replications

•DNA replication

1. Small animal viruses (simian virus 40, 5 kb) are good mammalian models for elongation (replication fork) but not for initiation.

2. Yeast (Saccharomyces cerevisiae): 1.4 X 107 bp in 16 chromosomes, 400 replicons, much simpler than mammalian system and can serve as a model system

3. Cell-free extract prepared from Xenopus (frog) eggs containing high concentration of replication proteins and can support in vitro replication.

Cell cycle When to replicate

•DNA replication

G1 preparing for DNA

replication (cell growth)S

DNA replicationG2

a short gap before mitosisM

mitosis and cell division

Cell cycle

Entry into the S-phase:Cyclins

Cyclin-dependent protein kinases (CDKs)

signaling

Iniation of multiple replicons

•DNA replication

1. Timing2. Order

1. Clusters of about 20-50 replicons initiate simultaneously at defined times throughout S-phase

• Early S-phase: euchromatin replication• Late S-phase: heterochromatin replication• Centromeric and telomeric DNA replicate last

2. Only initiate once per cell cycleLicensing factor: • required for initiation and inactivated after

use• Can only enter into nucleus when the

nuclear envelope dissolves at mitosis

Licensing factor

Initiation

Initiation: origin

1. Yeast replication origins (ARS- autonomously replicating sequences, enables the prokaryotic plasmids to replicate in yeast).Minimal sequence of ARS: 11 bp [A/T]TTTAT[A/G]TTT[A/T] (TATA box)Additional copies of the above sequence is required for optimal efficiency.

ORC (origin recognition complex) binds to ARS, upon activation by CDKs, ORC will open the DNA for replication.

- a complex of 6 ATPases- the functional equivalent of DnaA

Replication fork & elongation

•DNA replication

1. unwinding2. enzymes

Replication fork

Unwinding DNA from parental nucleosomes before replication : 50 bp/sec, helicases and RP-A

New nucleosomes are assembled to DNA from a mixture of old and newly synthesized histones after the fork passes.

Elongation: three different DNA polymerases are involved.

1. DNA pol : contains primase activity and synthesizes RNA primers for the leading strands and each lagging strand fragments. Continues elongation with DNA but is replaced by the other two polymerases quickly.

2. DNA pol : on the leading strand that replaces DNA pol . can synthesize long DNA

3. DNA pol : on the lagging strand that replaces DNA pol synthesized Okazaki fragments are very short (135 bp in SV40), reflecting the amount of DNA unwound from each nucleosome.

DNA Polymerase Switching

Nuclear Matrix

•DNA replication

A scaffold of insoluble protein fibers which acts as an organizational framework for nuclear processing, including DNA replication, transcription

Replication factories: all the replication enzymes, DNA associated with the replication forks in replication

BUdR labeling of DNA

Visualizing by immunoflurescence using BUdR antiboby

Telomere replication

•DNA replication

Solving the problem of lagging strand synthesis -- Chromosomal ends shortening

5’ 3’5’3’

3’ 5’3’5’

5’ 3’5’3’Parental DNA

Daughter DNAs

What about the ends (or telomeres) of linear chromosomes?

DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed, because DNA polymerases can only synthesize DNA only in the 5’ to 3’ direction and cannot initiate DNA synthesis

Big problem---If this gap is not filled, chromosomes would become shorter each round of replication!

Solution:

1. Eukaryotes have tandemly repeated sequences at the ends of their chromosomes - telomerestelomeres.

1. Telomerase (composed of protein and RNA complementary to the telomere repeat - This allows the telomerase to bind to the 3’ overhang) binds to the terminal telomere repeat and catalyzes the addition of of new repeats.

2. Compensates by lengthening the chromosome.3. Absence or mutation of telomerase activity results in chromosome

shortening and limited cell division.

Step 1 = Binding

Step 3 = Translocation

The binding-polymerization-

translocation cycle can occurs many times

This greatly lengthens one of the strands

The complementarystrand is made by primase, DNA polymerase and ligase

RNA primer

Step 2 = Polymerization

telomerase

•DNA replication

1. Contains a short RNA molecule as telomeric DNA synthesis template

2. Telomerase activity is repressed in the somatic cells of multicellular organism, resulting in a gradual shortening of the chromosomes with each cell generation, and ultimately cell death (related to cell aging)

3. The unlimited proliferative capacity of many cancer cells is associated with high telomerase activity.

Telomerase•DNA replication


Synthesis of telomeric DNA by telomerase

DNA polymerase control the fidelity of DNA replication

Proofreading refers to any mechanism for correcting errors in protein or nucleic acid synthesis that involves scrutiny of individual units after they have been added to the chain

Processive DNA polymerases have 3’5’ exonuclease activity

Supplemental 1

byE. coli polymerase

Proofreading

Supplemental 2

Conclusions

Cell division and DNA replication are coordinated processes

DNA replication is semiconservative

DNA synthesis occurs in the 5’3’ direction

DNA synthesis is semidiscontinuous:continuous on leading stranddiscontinuous on lagging strands

Lagging strand synthesis involves Okazaki fragments

Both procaryotes and eucaryotes contain multiple DNA polymerases which fulfil different but overlapping functions

DNA polymerases have proof-reading activity which corrects incorporation of incorrect nucleotides

Procaryotic chromosomes are replicated from a single ori, eucaryotic chromosomes from multiple ori

Replication can be uni- or bidirectional

How to clone a replicon

Conclusions

Proteins at the replication fork:

In bacteria replication starts at a single ori and terminates 180° opposite the ori where replication is arrested by replication fork traps - a physical barrier to replication fork progress

DNA PolIIIPrimase

Helicase SSBParental DNA

Leading strand


5’3’

3’5’

3’5’

+ DNA PolI+ Ligase

DNA replication proteins:DNA PolIIIDNA PolIDNA Ligase

Primase (DnaG)Helicase (DnaB)SSB

Replication terminationReplication fork traps opposite oriCTer sitesTus protein

Conclusions

I. Why do the properties of DNA polymerases yield priming and directionality problems?A. What are the solutions to these problems?B. How did Okazaki prove that Okazaki fragments are primed with RNA?C. Why might it be useful to prime Okazaki fragments with RNA?

II. What are the major components of the E. coli replisome and how do they work together to bring about semidiscontinuous DNA synthesis at a replication fork?

III. Components that act before DNA polymerase IIIA. How do hexameric helicases achieve strand separation?B. Why does the primosome consume ATP even when it is not synthesizing primers?

IV. DNA polymerase IIIA. What are the major modules within Pol III and what do they do?B. How are processivity and efficient recycling of Pol III at odds with one another?C. How do the clamp and the clamp loader work together to ensure both processivity and efficient recycling?

V. Components that act after DNA polymerase IIIA. What are the roles of DNA polymerase I and DNA ligase in finishing up the process of DNA replication?B. Why are topoisomerases necessary for DNA replication? Why do we specifically require topoisomerases that can reduce the linking number and why do we require type II topoisomerases?

VI. Initiation of replication - How do OriC and DnaA work together to initiate DNA replication?

DNA Replication - PW

Documents