Post on 11-May-2015
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-7
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-11
Interpreting the Data
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-12
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.
ReplicationReplication
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.
ReplicationReplication
5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
Leading strand synthesis continues in a 5’ to 3’ direction.
ReplicationReplication
3’ 5’ 5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
Okazaki fragment
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
5’
ReplicationReplication
5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Okazaki fragment
ReplicationReplication
5’
5’ 3’
5’
3’
3’
5’
3’
3’
5’ 5’ 3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
ReplicationReplication
3’
5’
3’
5’
5’ 3’
5’
3’
3’
5’ 5’ 3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
ReplicationReplication
5’
5’
3’ 3’
5’
3’
5’ 3’
5’
3’
3’
5’
Exonuclease enzymes remove RNA primers.
ReplicationReplication
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
Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)
DNA template is denatured with heat to separate strands.
C T T G A T CGC
3’5’
G ATCAA GCG
3’ 5’
Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)
DNA template is denatured with heat to separate strands.
C T T G A T CGC
3’5’
G ATCAA GCG
3’ 5’
Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)
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’
Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)
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’
Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)
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.
Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)
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
Study outline: