Central Dogma

Nucleic acid Lipi d Protei n Nucleic acid

Nucleic acid Nucleic acid + protein

Nucleic

Molecular Biology Course

DNA Replication

Molecular Biology Course

DNA Replication: An Overview

Replicons, semi-conservative, discontinous, RNA priming semi-

Bacterial DNA replication

Experimental system, initiation, unwinding, elongation, termination & segregation

Eukaryotic DNA replication

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

DNA replication

DNA Replication: An Overview

1. Replicons 2.semi-conservative mechanism 3.semi-discontinous replication 4.RNA priming

DNA replication

Replicons
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 Origin Replicon

ri c h aA bo xe s Ite ro Op ns er ato rs ite

rep gene

Dn

AT -

DNA replication

Prokaryotic genome: a single circular DNA = a single replicon Eukaryotic genome: multiple linear chromosomes & multiple replicons on each chromosome

Replication can be Uni- or Bidirectional

UNIDIRECTIONAL REPLICATION Origin 5 3 3 5

BIDIRECTIONAL REPLICATION Origin 5 3 3 5

DNA replication

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

Multiple eukaryotic replicons and replication bubbles

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 origins of replication in Eukaryotes

Are all origins created equal?

Rate of DNA synthesis and the need for multiple origins

Genome

Fork speed

S phase

Origins

Comment

E. coli

4.6 Mbp

30 kb/min

40 min

S longer than doubling time

Yeast

14 Mbp
(1 cm)

3 kb/min

20 min

330

S would last 80hr if only 1 ori

1 l culture = 4.1010 cells --> 400 000 km DNA synthesized (Earth-Moon distance)

Human

3 Gbp
(2 m)

3 kb/min

7h

>10 000 ?

S would last 1 year if 1 ori

2.1013 km DNA synthesized (2 light-years) during life time (1016 cell divisions)

Procaryotic (Bacterial) and Eucaryotic Chromosome Replication BACTERIAL CHROMOSOME

ori

ter

EUCARYOTIC CHROMOSOME

ori

ori

ori

DNA replication

eplication bubbles replication fork

DNA replication

Replication is Semiconservative

Proposed Models of DNA Replication

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

Alternative models of DNA replication :

Equilibrium density gradient centrifugation

DNA
1 4

1 5

 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 Stahls Experiment Semiconservative model of DNA replication

Interpreting the Data

After ~ two generations, DNA is of two types: light and half-heavy

This is consistent with only the semi-conservative model

After one generation, DNA is halfheavy

DNA replication

Replication is Semidiscontinuous

DNA replication

Semi-discontinuous replication

Ligation

Okazaki fragments

DNA Synthesis is Semidiscontinous

Lagging strand synthesis 5 3 5 3 3 5 Leading strand synthesis

Okazaki Fragment Synthesis

Leading strand synthesis

Primase synthesizes RNA primer

DNA PolIII extends primer into Okazaki fragment

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 semidiscontinuous on the lagging strand:

leading

strand

and

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

Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins: 5

SSB Proteins

Okazaki Fragments ATP 1 Polymerase III Lagging strand

Helicase + Initiator Proteins

3
primase Polymerase III
3
base pairs

5
RNA primer replaced by polymerase I

& gap is sealed by ligase

RNA Primer

Leading strand

DNA ligase seals the gaps between Okazaki fragments with a phosphodiester bond

DNA replication

Discovery of Okazaki fragments Evidence for semi-discontinuous replication

[3H] thymidine pulse-chase labeling experiment 1. Grow E. coli 2. 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 Synthesis Occurs in the 53 Direction

5 3 Incoming nuceolotide triphosphate 5 3 Nucleotide monophosphate added to chain with release of diphosphate 5 3 P P P P P P P P P P P P P OH 3 5

5PPP OH 3

OH3

PP OH 3

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 bp replicated 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 safeguards 1. Binding pocket of DNA polymerase clamps tightly around the base before catalysis occurs. Wobble pairs dont fit and so catalysis cant occur. 2. DNA polymerases have editing exonuclease activities that allow them to erase mistakes and try again 3. 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 enzyme DNA Pol II DNA repair DNA Pol III De novo synthesis of new DNA _______________________________________________ MAMMALIAN Enzyme Location Primary function

DNA Pol I ( ) Strand synthesis initiation Nucleus DNA Pol II ( ) DNA repair Nucleus DNA Pol III ( ) Strand extension Nucleus

Some DNApolymerases have 35 exonuclease activity

DNA polymerase

53 synthesis Incorrect nucleotide

Nucleotide misincorporation

Polymerase reversal and 35 exonuclease activity

Continued 53 synthesis

Proteins at the Replication Fork

Lagging strand (Okazaki fragment) 5 3 5 3 Leading strand Parental DNA 3 5

+ DNA PolI + Ligase

RNA primer

DNA polIII

Single-stranded binding (SSB) protein Primase DNA helicase

DNA Polymerase III

5 3 5 3
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 3OH termini, cannot initiate synthesis of new chains Catalyzes leading and lagging strand syntheses

3 5

DNA Pol III Catalyzes Phosphodiester Bond Formation in a DNA Chain

5PPP P P P P OH3

OH 3

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/sec processitivity 5 x 105 Processivity is due to sliding clamp of subunit:

DNA Polymerase I 1. 53 DNA chain synthesis:

103 kD Catalyzes DNA chain elongation by the formation of phosphodiester bonds Incoming dNTP is positioned for chain template nucleotide incorporation by H-bonding with

Can only extend chains from 3OH termini, cannot initiate synthesis of new chains Catalyzes lagging strand synthesis

2. 3 exonuclease activity:
A proofreading activity which results in the removal of improperly-paired nucleotide

DNA Polymerase I (continued) 3. 5 exonuclease activity:

Excision of RNA primer from Okazaki fragment during lagging strand synthesis
5
Okazaki fragment

RNA primer

Nick

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 3OH and a 5phosphate end is generated DNA ligase catalyzes phosphodiester bondformation on nick: 5 3 P 5 HO 3 3 5

5 3

3 5

Primase (DnaG)
60 kD Intiates Okazaki fragment synthesis from a single-stranded DNA template:

Primase

RNA primer

After addition of 10-12 ribonucleotides, primase is displaced by DNA PolIII which synthesizes DNA from the 3OH group on RNA primer Complexed with helicase in lagging strand

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 SSB ssDNA

>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:

Proteins at the Replication Fork

Lagging strand (Okazaki fragment) 5 3 5 3 Leading strand Parental DNA 3 5

+ DNA PolI + Ligase

RNA primer

DNA polIII

Single-stranded binding (SSB) protein Primase DNA helicase

DNA replication

Bacterial DNA replication

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

BACTERIAL REPLICATION
DNA synthesis begins at a site termed the origin of replication
Each bacterial chromosome has only one

Synthesis of DNA proceeds bidirectionally around the bacterial chromosome

eventually meeting at the opposite side of the bacterial chromosome
Where replication ends

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 I II III Polymerization (5-3) Yes Yes Yes Exonuclease (3-5) Exonuclease (5-3) #Copies Yes Yes Yes Yes No No 400 ? 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 in E. coli : 1. 2. Two replication forks result in a theta-like ( ) structure. As strands separate, positive supercoils form elsewhere in the molecule. Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate.

3.

Rolling circle model replication : 1. 2. 3.

of

DNA

Common in several bacteriophages including . Begins with a nick at the origin of replication. 5 end of the molecule is displaced and acts as primer for DNA synthesis. Can result in a DNA molecule many multiples of the genome length (and make multiple copies quickly). During viral assembly the DNA is cut into individual viral chromosomes.

4.

5.

DNA replication

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 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 region DnaA 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.

DNA Polymerase Cannot Initiate new Strands

Unable to covalently link the 2 individual nucleotides together

Fig. 11.9a(TE Art)

3 5 5 3 5

Able to covalently link together

ri P

er

3 5

Synthesis and replacement of RNA primers during DNA replication

Initiation 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

DNA replication

Unwinding
Positive supercoiling: caused by removal of helical turns at the replication fork. Resolved by a type II topoisomerase called DNA gyrase

Topoisomerase at the Replication Fork

DNA replication

Elongation

Three Dimensional view of Replication Fork

Direction of synthesis of leading strand

Direction of synthesis Of lagging strand

Direction of fork movement

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 rate 3. Both polymerases contain an -subunit--polymerase -subunit---35 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 Hydrolyis 2. Single-stranded DNA binding proteins (SSB) - Bind and stabilize the DNA in a single stranded conformation after the melting by helicases 3. Primosome - Synthesizes RNA primers for the lagging strand 4. DNA Polymerase III - The replicase 5. Type II Topoisomerase (Gyrase) - Relaxes postively supercoiled DNA that forms ahead of the replication fork. Decatenates the final product 6. DNA Polymerase I - Replaces RNA primers with DNA by nick translation 7. DNA Ligase - Joins the Okazaki fragments

Elongation: lagging strand replication

Polymerase III holoenzyme (DNA pol III) DNA pol I (53 exonulclease activity)

DNA pol I (53 polymerase activity)

DNA ligase

Directionality of the DNA strands at a replication fork

Fork movement Lagging strand Leading strand

DNA elongation :

DNA replication

Termination and Segregation

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

ter

Replication Termination of the Bacterial Chromosome

BIDIRECTIONAL REPLICATION Origin 5 3 3 5

ori

ter

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

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

Replication Termination of the Bacterial Chromosome

Chromosome

TerB

TerA

Replication Termination of the Bacterial Chromosome

Ter sites are binding sites for the Tus protein Tus: 35.8 kD DNA binding at Ter Monomer
Tus DNA Replication fork arrested in polar manner

Ter

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

Segregation

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

odel of replication in E. coli

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

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

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

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
1. Experimental system 2. 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. 2. 3. 4. Cells must be large enough, and the environment favorable. Cell will not enter the mitotic phase unless all the DNA has replicated. Chromosomes also must be attached to the mitotic spindle for mitosis to complete. 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. 2. 3. 4. 5. Polymerase (alpha): nuclear, DNA replication, no proofreading Polymerase (beta): nuclear, DNA repair, no proofreading Polymerase (gamma): mitochondria, DNA repl., proofreading Polymerase (delta): nuclear, DNA replication, proofreading 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 - the eukaryotic replicase Pol /primase - contains both primase and DNA polymerase Activities 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

DNA replication

In vitro experimental systems

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

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.

DNA replication

Cell cycle
When to replicate

Cell cycle
G1 preparing for DNA replication (cell growth) S DNA replication G2 a short gap before mitosis M mitosis and cell division Entry into the S-phase: Cyclins
Cyclin-dependent protein kinases (CDKs)

signaling

DNA replication

Iniation of multiple replicons

1. Timing 2. Order

1. Clusters of about 20-50 replicons initiate simultaneously at defined times throughout Sphase Early S-phase: euchromatin replication Late S-phase: heterochromatin replication Centromeric and telomeric DNA replicate last

2. Only initiate once per cell cycle Licensing factor: required for initiation and inactivated after use Can only enter into nucleus when the nuclear envelope dissolves at mitosis

Initiation Licensing factor

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

DNA replication

Replication fork & elongation

1. unwinding 2. 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

DNA replication

Nuclear Matrix
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

DNA replication

Telomere replication
Solving the problem of lagging strand synthesis -- Chromosomal ends shortening
Parental DNA 5 3
3 5 5 5 3 3 5 3

Daughter DNAs

5 3

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. 1. 2. 3. Eukaryotes have tandemly repeated sequences at the ends of their chromosomes - telomeres. telomeres 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. Compensates by lengthening the chromosome. Absence or mutation of telomerase activity results in chromosome shortening and limited cell division.

Step 1 = Binding

The bindingpolymerizationtranslocation cycle can occurs many times This greatly lengthens one of the strands

Step 2 = Polymerization

Step 3 = Translocation

The complementary strand is made by primase, DNA polymerase and ligase

RNA primer

DNA replication

telomerase

DNA replication

Telomerase

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.

Synthesis of telomeric DNA by telomerase

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

Supplemental 1 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 exonuclease activity polymerases have 35

Supplemental 2

Proofreading by E. coli polymerase

Conclusions
Cell division and DNA replication are coordinated processes DNA replication is semiconservative DNA synthesis occurs in the 53 direction DNA synthesis is semidiscontinuous: continuous on leading strand discontinuous on lagging strands Lagging strand synthesis involves Okazaki fragments

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

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

Proteins at the replication fork:

Lagging strand (Okazaki fragment) 5 3 5 3 Leading strand DNA PolIII Primase Helicase SSB Parental DNA 3 5
+ DNA PolI + Ligase

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

Conclusions
DNA replication proteins: DNA PolIII DNA PolI DNA Ligase Primase (DnaG) Helicase (DnaB) SSB

Replication termination Replication fork traps opposite oriC Ter sites Tus protein

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 III A. How do hexameric helicases achieve strand separation? B. Why does the primosome consume ATP even when it is not synthesizing primers? IV. DNA polymerase III A. 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 III A. 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?