Dna replication and pcr

Genetics: Analysis and Genetics: Analysis and PrinciplesPrinciples

CHAPTER 11CHAPTER 11

DNA replicationDNA replication

By Robert J. BrookerBy Robert J. Brooker

DNA Structure Helps DNA Structure Helps Explain How It DuplicatesExplain How It Duplicates

DNA is two nucleotide strands held DNA is two nucleotide strands held

together by hydrogen bondstogether by hydrogen bonds

Hydrogen bonds between two strands Hydrogen bonds between two strands

are easily brokenare easily broken

Each single strand then serves as Each single strand then serves as

template for new strandtemplate for new strand

DNA DNA ReplicationReplication

Each old strand Each old strand

stays intact stays intact

Each new DNA Each new DNA

molecule is half molecule is half

“old” and half “old” and half

“new”“new”

Fig. 1-7, p.212

DNA is a double helixDNA is a double helixP

A

P C

P

G

P T

P C

P

G

P

A

PC

PT

G

P

PC

P

A sugar and phosphate “backbone” connects nucleotides in a chain.

P

G

P

Two nucleotide chains together wind into a helix.

DNA strands are antiparallel.

DNA has directionality.

5’

3’

3’

5’

Hydrogen bonds between paired bases hold the two DNA strands together.

DNA Melting Temperature

Orientation of DNAOrientation of DNA

The directionality of a DNA strand is due to the orientation of The directionality of a DNA strand is due to the orientation of the phosphate-sugar backbone.the phosphate-sugar backbone.

The carbon atoms on the sugar ring are numbered for reference. The 5’ and 3’ hydroxyl groups (highlighted on the left) are used to attach phosphate groups.

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

• Both parental strands stay together after DNA replicationBoth parental strands stay together after DNA replication

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

daughter strand following replicationdaughter strand following replication

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

following replicationfollowing replication

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Experiment : Which Model of Experiment : Which Model of DNA Replication is Correct?DNA Replication is Correct?

11-5

Figure 11.211-6

In 1958, Matthew Meselson and Franklin Stahl In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these modelsdevised a method to investigate these models They found a way to experimentally distinguish They found a way to experimentally distinguish

between daughter and parental strandsbetween daughter and parental strands

Their experiment can be summarized as suchTheir experiment can be summarized as such Grow Grow E. coliE. coli in the presence of in the presence of 1515N (a heavy isotope of N (a heavy isotope of

Nitrogen) for many generationsNitrogen) for many generations• The population of cells had heavy-labeled DNAThe population of cells had heavy-labeled DNA

Switch Switch E. coliE. coli to medium containing only to medium containing only 1414N (a light N (a light isotope of Nitrogen)isotope of Nitrogen)

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

a CsCl gradienta CsCl gradient

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The HypothesisThe Hypothesis

This experiment aims to determine which of the This experiment aims to determine which of the three models of DNA replication is correctthree models of DNA replication is correct

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Testing the HypothesisT Refer to Figure 11.3

11-8

11-9Figure 11.3

Figure 11.311-10

The Data


Interpreting the Data


After one generation, DNA is “half-heavy”

This is consistent with both semi-conservative and dispersive models

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

This is consistent with only the semi-conservative model

Meselson-Stahl Experiment

Conclusion: Replication is semiconservative.

Important rules about DNA/RNA

1. The base sequence is always shown in 5’ to 3’ direction

Thus:: AGCTCCGCTA means 5’ AGCTCCGCTA 3’

The complementary strand of 5’ AGCTCCGCTA 3’ Is then 5’ TAGCGGAGCT 3’ !!!!!!!!!

Replicatoin can’t just start:

1. All DNA polymerases need a primer1. All DNA polymerases need a primer

2. The primer can be a piece of RNA or DNA2. The primer can be a piece of RNA or DNA

3. It must be “base-paired” with the “template” and with 3. It must be “base-paired” with the “template” and with 3’OH3’OH

Thus:Thus:

3’ 5’5’ 3’

Template strandTemplate strand

primerprimer

Synthesis directionSynthesis direction

A Closer Look at A Closer Look at Strand AssemblyStrand Assembly

Energy for strand Energy for strand assembly is assembly is provided by provided by removal of two removal of two phosphate groups phosphate groups from free from free nucleotidesnucleotides

newlyformingDNAstrand

one parent DNA strand

““Proof-reading” is essential at DNA replicationProof-reading” is essential at DNA replication

•What is proof-reading?What is proof-reading?

1. If there is a wrong base built in, then there is no base 1. If there is a wrong base built in, then there is no base paring possible.paring possible.

2. The DNA polymerase can’t continue on building in the 2. The DNA polymerase can’t continue on building in the next base.next base.

3. The DNA polymerase removes the “wrong” base3. The DNA polymerase removes the “wrong” base and starts overand starts over

Klenow Fragment (of pol I)(The proof-reading activity)

DNA pol IDNA pol I Composed of a single polypeptideComposed of a single polypeptide Removes the RNA primers and replaces them with DNA Removes the RNA primers and replaces them with DNA

DNA pol IIIDNA pol III Responsible for most of the DNA replicationResponsible for most of the DNA replication Composed of 10 different subunits (Table 11.2)Composed of 10 different subunits (Table 11.2)

• The The subunit synthesizes DNA subunit synthesizes DNA• The other 9 fulfill other functionsThe other 9 fulfill other functions

The complex of all 10 is referred to as the The complex of all 10 is referred to as the DNA pol III DNA pol III holoenzymeholoenzyme

DNA PolymerasesDNA Polymerases

There are 3 others pol II, IV and V, There are 3 others pol II, IV and V, involved in repair (no details)involved in repair (no details)

Eukaryotes have ~ 15 DNA polymerases !Eukaryotes have ~ 15 DNA polymerases !

Each polymerase has its own function!Each polymerase has its own function! DNA synthesis during genome replication (cell DNA synthesis during genome replication (cell

division) division) Other are involved in DNA damage repairOther are involved in DNA damage repair

Bacterial DNA polymerases may vary in their Bacterial DNA polymerases may vary in their subunit compositionsubunit composition However, they have the same type of catalytic subunitHowever, they have the same type of catalytic subunit

Structure resembles a human right hand

Template DNA thread through the palm;

Thumb and fingers wrapped around the DNA

The figure presents an overview of the process of bacterial The figure presents an overview of the process of bacterial chromosome replicationchromosome replication

DNA synthesis begins at a site termed the DNA synthesis begins at a site termed the origin of replicationorigin of replication• Each bacterial chromosome has only oneEach bacterial chromosome has only one

Synthesis of DNA proceeds Synthesis of DNA proceeds bidirectionallybidirectionally around the bacterial around the bacterial chromosomechromosome

The replication forks eventually meet at the opposite side of the The replication forks eventually meet at the opposite side of the bacterial chromosomebacterial chromosome

• This ends replicationThis ends replication

BACTERIAL DNA REPLICATIONBACTERIAL DNA REPLICATION

The origin of replication in The origin of replication in E. coliE. coli is termed is termed oriCoriC oriorigin of gin of CChromosomal replicationhromosomal replication

Three types of DNA sequences in Three types of DNA sequences in oriCoriC are are functionally significantfunctionally significant AT-rich regionAT-rich region DnaA boxesDnaA boxes GATC methylation sitesGATC methylation sites

Initiation of ReplicationInitiation of Replication

Proteins involved in E. coli replication

Terms to be known

Why the discontinuous additions? Nucleotides can only be joined to an exposed —OH group that is attached to the 3’ carbon of a growing strand.

Strand AssemblyStrand Assembly

Continuous and Discontinuous Continuous and Discontinuous AssemblyAssembly

Binding proteins prevent single strands from rewinding.

ReplicationReplication

Helicase protein binds to DNA sequences called origins and unwinds DNA strands.

5’ 3’

5’

3’

Primase protein makes a short segment of RNA complementary to the DNA, a primer.

3’ 5’

5’ 3’

ReplicationReplicationOverall direction

of replication 5’ 3’

5’

3’

5’

3’

3’ 5’

DNA polymerase enzyme adds DNA nucleotides to the RNA primer.


DNA polymerase enzyme adds DNA nucleotides to the RNA primer.

5’

5’

Overall directionof replication

5’

3’

5’

3’

3’

3’

DNA polymerase proofreads bases added and replaces incorrect nucleotides.


5’

5’ 3’

5’

3’

3’

5’

3’Overall directionof replication

Leading strand synthesis continues in a 5’ to 3’ direction.


3’ 5’ 5’

5’ 3’

5’

3’

3’

5’


Okazaki fragment


Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

5’


5’

5’ 3’

5’

3’

3’

5’


3’



Okazaki fragment


5’

5’ 3’

5’

3’

3’

5’

3’

3’

5’ 5’ 3’




3’

5’

3’

5’

5’ 3’

5’

3’

3’

5’ 5’ 3’




5’

5’

3’ 3’

5’

3’

5’ 3’

5’

3’

3’

5’

Exonuclease enzymes remove RNA primers.


Exonuclease enzymes remove RNA primers.

Ligase forms bonds between sugar-phosphate backbone.

3’

5’

3’

5’ 3’

5’

3’

3’

5’

Enzymes in ReplicationEnzymes in Replication

Enzymes (Enzymes (HelicasesHelicases) unwind the two ) unwind the two

strandsstrands

DNA polymeraseDNA polymerase needed for the synthesis needed for the synthesis

of complementary strand of complementary strand

DNA ligaseDNA ligase joins pieces of the lagging joins pieces of the lagging

strand togetherstrand together

Enzymes in DNA replicationEnzymes in DNA replication

Helicase unwinds parental double helix

Binding proteinsstabilize separatestrands

DNA polymerase binds nucleotides to form new strands

Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone

Primase adds short primer to template strand

Exonuclease removesRNA primer and inserts the correct bases

DNA replication mistakesDNA replication mistakes

The most errors in DNA sequence occur during The most errors in DNA sequence occur during

replication.replication.

Reparation takes place after replication is Reparation takes place after replication is

finishedfinished

DNA polymerases can get the right sequence DNA polymerases can get the right sequence

from the complementary strand and repair, from the complementary strand and repair,

along with DNA ligase, the wrong bases. along with DNA ligase, the wrong bases.

Eukaryotic DNA replicationEukaryotic DNA replication is not as well is not as well understood as bacterial replicationunderstood as bacterial replication

The two processes do have extensive similarities,The two processes do have extensive similarities,• The bacterial enzymes described before have also The bacterial enzymes described before have also

been found in eukaryotesbeen found in eukaryotes

Nevertheless, DNA replication in eukaryotes is more Nevertheless, DNA replication in eukaryotes is more complexcomplex• Large linear chromosomesLarge linear chromosomes• Tight packaging within nucleosomesTight packaging within nucleosomes• More complicated cell cycle regulationMore complicated cell cycle regulation

EUKARYOTIC DNA REPLICATIONEUKARYOTIC DNA REPLICATION

Multiple origins of replication in eukaryotes

Origins of replication in Saccharomyces cerevisiae are termed ARS elements (Autonomously Replicating Sequence)

They are 100-150 bp in lengthThey have a high percentage of A and TThey have three or four copies of a specific

sequence

The replication rate is similar to E. coli

Linear eukaryotic chromosomes have telomeres at Linear eukaryotic chromosomes have telomeres at both endsboth ends

The term The term telomeretelomere refers to the complex of refers to the complex of telomeric DNA sequences and bound proteinstelomeric DNA sequences and bound proteins

Telomeres and DNA Telomeres and DNA ReplicationReplication

Telomeric sequences consist ofTelomeric sequences consist of Moderately repetitive tandem arraysModerately repetitive tandem arrays 3’ overhang that is 12-16 nucleotides long3’ overhang that is 12-16 nucleotides long

Telomeric sequence typically for ‘vertebrates’: Telomeric sequence typically for ‘vertebrates’: TTAGGGTTAGGG

See: http://telomerase.asu.edu/sequences.htmlSee: http://telomerase.asu.edu/sequences.html

DNA polymerases possess two unusual DNA polymerases possess two unusual featuresfeatures 1. They synthesize DNA only in the 5’ to 3’ direction1. They synthesize DNA only in the 5’ to 3’ direction 2. They cannot initiate DNA synthesis2. They cannot initiate DNA synthesis

These two features pose a problem at the 3’ end of These two features pose a problem at the 3’ end of linear chromosomes-the end of the strand cannot linear chromosomes-the end of the strand cannot be replicated!be replicated!

That is why the ‘normal’ differentiated cells have That is why the ‘normal’ differentiated cells have linear chromosomes that become shorter and linear chromosomes that become shorter and shorter and thus these cells have an ending life.shorter and thus these cells have an ending life.

Only Only cancercancer and and stamstam cells can solve this problem by cells can solve this problem by expression of a expression of a telomerasetelomerase enzyme. enzyme.

The The telomerasetelomerase contains contains proteinprotein and and RNARNA The RNA is complementary to the DNA sequence of the The RNA is complementary to the DNA sequence of the

telomere ‘repeat’ sequence.telomere ‘repeat’ sequence.• Therefore, it can bind to the 3’-end of the chromosomes.Therefore, it can bind to the 3’-end of the chromosomes.

Next slide will give more explinationNext slide will give more explination

Step 1 = Binding

Step 3 = Translocation

The binding-polymerization-

translocation cycle can occurs many times

This greatly lengthens one of the strands

RNA primer

Step 2 = Polymerization

The end is now lengthened

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Is a method in which multiple repetitions of DNA replication are performed in a test tube.

Mix in test tube:

DNA template DNA to be amplified

Primers one complementary to each strand

Nucleotides dATP,d GTP, dCTP, and dTTP

DNA polymerase heat stable form from thermophilic bacteria


DNA template is denatured with heat to separate strands.

C T T G A T CGC

3’5’

G ATCAA GCG

3’ 5’


C T T G A T CGC

3’5’

G ATCAA GCG

3’ 5’

Each DNA primer anneals, binding to its complementary sequence on the template DNA

C T T

GCG

5’5’

3’3’


DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer.

C T T G A T CGC

3’5’

G ATCAA GCG

3’ 5’

C T T

GCG

5’5’3’

C G CG A T

G A A C T A

3’


Denaturation

Each DNA primer anneals, bindingto its complementary sequenceon the template DNA

DNA template is denatured with high heat to separate strands.

Annealing

Extension DNA polymerase creates a new strand of DNA complementaryto the template DNA starting from the primer.

Multiple rounds of denaturation-annealing-extension areperformed to create many copies of the template DNA between the two primer sequences.


After 35 cycles a single molecule will be amplified 235 times

= ± 50.000.000.000 x

Visualization of PCR products

agarose gel-electrophoresis of DNATop

Bottom

Slab of agarose gel (ethidium bromide staining)

Positive pole

Negative pole

DNA is Negatively charged

Small molecules dissolve faster

separation based on size

Structural Overview of DNA Replication Existing DNA strands act as templates for the synthesis of new strandsThree different models were proposed that described the net result of DNA replication

Bacterial DNA Replication Bacterial chromosomes contain a single origin of replicationReplication is initiated by the binding of DnaA protein to the origin of replicationSeveral proteins are required for DNA replication at the replication forkDNA polymerase III is a processive enzyme that uses deoxyribonucleoside triphosphates Replication is terminated when the replication forks meet at the terminus sequencesCertain enzymes of DNA replication bind to each other to form a complexThe fidelity of DNA replication is ensured by proofreading mechanismsBacterial DNA replication is coordinated with cell divisionDNA replication can be studied in vitroThe isolation of mutants has been instrumental in our understanding of DNA replication

Eukaryotic DNA replication Initiation occurs at multiple origins of replication on linear eukaryotic chromosomesEukaryotes contain several different DNA polymerasesThe ends of eukaryotic chromosomes are replicated by telomerase

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Dna replication and pcr

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