DNA Replication Biology 12. 6-9 600Cells can contain 6-9 feet of DNA. If all the DNA in your body...
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Transcript of DNA Replication Biology 12. 6-9 600Cells can contain 6-9 feet of DNA. If all the DNA in your body...
![Page 1: DNA Replication Biology 12. 6-9 600Cells can contain 6-9 feet of DNA. If all the DNA in your body was put end to end, it would reach to the sun and back.](https://reader035.fdocuments.in/reader035/viewer/2022070415/5697c02c1a28abf838cd8e92/html5/thumbnails/1.jpg)
DNA Replication
Biology 12
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•Cells can contain 6-96-9 feet of DNA. If all the DNA in your body was put end to end, it would reach to the sun and back over 600 600 times.
•DNA in all humans is 99.9 99.9 percent identical. It is about one tenth of one percent that makes us all unique, or about 3 million nucleotides difference.
•DNA can store 2525 gigabytes of information per inch and is the most efficient storage system known to human. So, humans are better than computers!!
•In an average meal, you eat approximately 55,000,00055,000,000 cells or between 63,000 to 93,00063,000 to 93,000 miles of DNA.
•It would take a person typing 60 words per minute, eight hours a day, around 5050 years to type the human genome.
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DNA is composed of units called NUCLEOTIDESNUCLEOTIDES, which are composed of three sub-molecules:
1. Pentose SugarPentose Sugar (deoxyribose)2. PhosphatePhosphate3. Nitrogen BaseNitrogen Base (purine or pyrimidine)
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4
Two Kinds of Bases in DNA
PyrimidinesPyrimidines are are singlesingle ring bases ring bases..
PurinesPurines are are doubledouble ring ring bases.bases.
C
C
C
C
N
N
OO
N
C
C
C
C
N
N
N
N
N
C
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DNA is composed of two complimentarycomplimentary strands of nucleotides joined by hydrogen bondshydrogen bonds::
Adenine Adenine with Thymine Thymine (A-T or T-A)They join with 22 hydrogen bonds
Cytosine Cytosine with Guanine Guanine (C-G or G-C)They join with 33 hydrogen bonds
DNA twists into a double helix double helix
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6
Chargraff’s Rule:Chargraff’s Rule:
• Adenine and Thymine always join together
A T
• Cytosine and Guanine always join together
C G
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The bases always pair up in the same way to form base pairs.
Adenine forms a bond with Thymine
and Cytosine bonds with Guanine
Bonding 1 10
Adenine Thymine
Cytosine Guanine
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PO4
PO4
PO4
thymine
PO4
PO4
PO4
PO4
adenine
cytosine
PO4
guanine
Bonding 2 11
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Replication
Copying the genetic material is REPLICATION.
Replication occurs prior to
cell division, because the new, daughter cell will also need a complete copy of cellular DNA.
Replication
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DNA Replication Problem Solving
On the surface…. The replication of DNA is pretty simple. Just unzip, plug in the the spare parts by complementary base pairing, and stitch up the new backbone.
There are a lot of irritating details and problems with this process.
The solutions to these problems involve a list of vital enzymes.
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DNA Replication Complexities:
DNA is a very stable molecule but its stability depends on its double stranded nature. Single stranded DNA is vulnerable to a number of kinds of damage. So…..to take a huge DNA molecule and seperate its two strands for its entire length....is a very bad idea.
So how can long DNA molecules be replicated without making them single stranded for long periods of time?
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DNA is very long. VERY long. How can a very long DNA molecule get itself replicated in a relatively short period of time?
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The primary DNA replication enzyme, DNA Polymerase, is highly specific in a number of ways. One of those specificities is that it can only add new nucleotides to an already existing growing strand of nucleotides. This is a problem because it means that DNA polymerase is not capable of actually starting the process itself. How does replication get started?
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DNA polymerase is also highly specific in that it can only build new polynucleotide strands in the 5' to 3' direction--new strands must always run from 5' to 3'. But the two strands of DNA are antiparallel to each other--one runs 3' to 5', the other from 5' to 3'. Both sides have to be replicated. How is this puzzle solved in DNA replication? What's the name of the scientist credited with this discovery?
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DNA is double stranded, and the two strands twist around each other. These two strands need to be pulled apart, thus tightening the twist between Origin points. This would lead to accidental breakage of the polynucleotide strands as the twists got compressed into smaller and smaller lengths. How is this problem avoided in DNA replication? What is the name of the enzyme needed to solve this problem (it has several; any of them will do)?
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Finally, the solutions to the previous problems leave us with an embarrassing problem: single stranded breaks in the polynucleotides of our new DNA molecules. How are these breaks repaired? Again, you'll need to identify the enzyme involved.
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DNA Replication Overview
1. Enzyme breaks weak hydrogen bonds
2. DNA strands open up
3. Free nucleotides (from our food) fill in the open side (free nucleotides are a significant component of the nucleoplasm in any cell.
Using complimentary
base pairing
End Result: 2 identical DNA strands
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How Does DNA Replicate? Three Hypotheses:
ConservativeConservative Semi-Semi-ConservativeConservative
DispersiveDispersive2 strands of the parent stay together, daughter gets new two strands
2 strands of parent separate, daughter gets 1 strand of DNA from parent
New DNA is made of a random Mixture of parent and daughter DNA
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Meselson-Stahl Meselson-Stahl
Discovered the ‘semi-conservative’ model
Experimented with bacteria
Replication fork (site of replication)
Figure 10.6
Parental (old)DNA molecule
Daughter(new) strand
DaughterDNA molecule(double helices)
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Semi-Conservative Replication
Origin of name: Original parent
strands conserved BUT are not still attached together
End Result: Each new daughter
DNA strand is ½ “old” and ½ “new”
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Three Steps In DNA Replication
1. Initiation – Replication begins at a location on the double helix known as “oriC” to which a certain initiator proteins bind and trigger unwinding. Enzymes known as helicases unwind the double helix by breaking the hydrogen bonds between complimentary base pairs, while other proteins keep the single strands from rejoining. The topoisomerase proteins surround the unzipping strand and relax the twisting that migh damage the unwinding DNA.
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2. Elongation With the primer as the starting point for the leading
strand, a new DNA strand grows one base at a time. The old (existing) strand is the template for the new strand. The enzyme DNA polymerase controls elongation, which can only occur in the leading direction.
The lagging strand unwinds in small sections that DNA polymerase replicates in the leading direction. The resulting “Okazaki fragments” can contain between 100 to 200 bases. The fragments terminate in an RNA primer that is later removed so that enzymes stitch the back together into one long strand.
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3. Termination
After the elongation is completed, two new double helices have replaced the original one.
During termination the last primer must be removed from the end of the lagging strand.
Enzymes proofread the new double helix and remove mispaired bases.
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- Enzyme breaks the weak - Enzyme breaks the weak hhydrogen bondsydrogen bonds
- Splitting the parent DNA strandSplitting the parent DNA strand
- Leaving two separated strandsLeaving two separated strands
Step #1 Separating DNA StrandsGyraseRelieves tension by unwindingHelicaseBreaks hydrogen bondsUnzips the DNATerminates at fork
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Separating DNA StrandsSSBs (Single-stranded binding proteins)
Bind to the exposed DNAKeep from “annealing”
• Reattaching with complimentary base pair
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The two sides of the molecule are separated for a short distance. Since DNA is most stable (and least vulnerable to damage) in itsdouble stranded configuration,as little of it as possible will besingle stranded at once.
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DNA ReplicationDNA Replication
Priming:Priming:
1.1. RNA primersRNA primers: before new DNA strands can form, there must be small pre-existing
primers (RNA)primers (RNA) present to start the addition of new nucleotides ((DNA PolymeraseDNA Polymerase)).
2.2. PrimasePrimase: enzyme that polymerizes (synthesizes) the RNA Primer.
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DNA polymerase performs only one job, following the complementary base pairing rule. It adds the new free nucleotides in the new strand of a replicating DNA molecule.
This also means that DNA polymerase cannot actually start the processof replication.
An enzyme called primase (an RNA polymerase) actually begins the replication process. It builds a short piece of RNA called a primer. This primer is later removed by RNAse H and replaced by DNA nucleotides.
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- Free nucleotides with the assistance of a protein Free nucleotides with the assistance of a protein called called DNA polymeraseDNA polymerase, attach to original parent , attach to original parent DNA strand which serves as a DNA strand which serves as a templatetemplate..
- Nucleotides in the new strand are selected using Nucleotides in the new strand are selected using complimentary base pairingcomplimentary base pairing
- A with T and C with GA with T and C with G
Step #2 Building Complimentary Strands
Replication proceeds alongBoth sides of the replication fork.
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DNA polymerase is only capable of building a new strand from the 5’ end to the 3’ end. This is a problem because the two sides of the DNA are antiparallel.
One side of the new strand (called the leading strand) can be directly and continuously constructed from its 5’ end to its 3’ end.
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DNA ReplicationDNA Replication
Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:
1.1. DNA PolymeraseDNA Polymerase: with a RNA primerRNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the synthesis of a new DNA strand in the
55’’ to 3to 3’’ direction direction.
RNARNAPrimerPrimerDNA PolymeraseDNA Polymerase
NucleotideNucleotide
5’
5’ 3’
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DNA ReplicationDNA Replication
2.2. Leading StrandLeading Strand: synthesized as a single polymersingle polymer in the 55’’ to 3 to 3’’
directiondirection.
RNARNAPrimerPrimerDNA PolymeraseDNA PolymeraseNucleotidesNucleotides
3’5’
5’
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Figure 5
III
Parent strand
Replication fork
Single-stranded binding proteins
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The other strand (called the lagging strand) must be constructed in short segments, built backwards.
These short strands of new DNA are called Okasaki fragments after the man who discovered them.
The Okasaki fragments will eventually be connected by an enzyme named DNA ligase. DNA ligase specializes in healing single stranded nicks in DNA. It simply seals the bond between one nucleotide and the neighboring nucleotide.
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DNA ReplicationDNA Replication
3.3. Lagging StrandLagging Strand: also synthesized in the 55’’ to 3 to 3’’ direction direction, but
discontinuouslydiscontinuously against overall direction of replication.
RNA PrimerRNA Primer
Leading StrandLeading Strand
DNA PolymeraseDNA Polymerase
5’
5’
3’
3’
Lagging StrandLagging Strand
5’
5’
3’
3’
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DNA ReplicationDNA Replication
4.4. Okazaki FragmentsOkazaki Fragments: series of short segments on the lagging strand.lagging strand.
Lagging Strand
RNARNAPrimerPrimer
DNADNAPolymerasePolymerase
3’
3’
5’
5’
Okazaki FragmentOkazaki Fragment
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DNA ReplicationDNA Replication
5.5. DNA ligaseDNA ligase: a linking enzyme that catalyzes the formation of a
covalent bond from the 3 3’’ to 5 to 5’’ end end of joining stands.
Example: joining two Okazaki fragments together.Example: joining two Okazaki fragments together.
Lagging Strand
Okazaki Fragment 2Okazaki Fragment 2
DNA ligaseDNA ligase
Okazaki Fragment 1Okazaki Fragment 1
5’
5’
3’
3’
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DNA ligase Joins Okazaki fragments together
Completes backboneLagging strand only!
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Building Complimentary Strands
Leading strand Built continuously From parent 3’ end toward replication
fork
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Building Complimentary Strands
Lagging strand Built in short Okazaki fragments
between RNA primersDiscontinuousFrom parent 3’ end away from
replication fork
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III
Parent strand
Replication fork
Single-stranded binding proteins
Okazaki fragments
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Step 1
Step 2
Step 3
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DNA is extremely long. It would take a very long time to replicate the whole molecule from end to end using only a single replication fork.
Each of these long molecules have many sites called Origins. This forms a configuration called a replication bubble. Each replication bubble actually has two forks, one on each end of the bubble and travelling in opposite directions.
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Fork vs. Bubble
Replication Bubble: Begins at multiple
sites Produces multiple
forks Proceeds in both
directions Result:
Faster replication
Figure 10.8
Origin ofreplication
Origin ofreplication
Origin ofreplication
Parental strand
Daughter strand
Bubble
Two daughter DNA molecules
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DNA cannot be double stranded and not twist. Unless there is a way to relax the twists between replication bubbles, the helical twists would compress putting increasing stress on the molecule causing random breaking and damage. This problem is solved by the creation of single stranded breaks (swivels) between origin sites.These nicks are made by an enzyme calledDNA topoisomerase (previously called “unwindase”, then “swivelase”.After the replication bubbles meet, ‘the single strands arehealed by DNA Ligase.
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Proofreading
DNA polymerase I and III Check for proper base
pairing If mistake found:
Removes Replace with proper
nucleotide
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- Results in two identical daughter DNA strandsResults in two identical daughter DNA strands
- ½ new and ½ old½ new and ½ old
End Result
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Leading vs. Lagging
Leading Strand
Lagging Strand
Toward replication fork
Direction complimentar
y strand is built
Away from replication fork
Yes Built continuously?
No(Okazaki
fragments)
Only once Number of times RNA primers are
needed
Multiple times (as replication fork moves)
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Leading vs. Lagging
Lagging ONLYDNA ligase completes backbone
bonds
Both:Built from parent 3’ to 5’ end
• From daughter 5’ to 3’ endUse DNA polymerase III & I
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Leading vs. Lagging
Why do we need leading and lagging strands?
DNA polymerase III can only build from parent 3’ end
DNA runs antiparallel When “unzipping”:
Leading: 3’ end IS available Lagging: 3’ end IS NOT available
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Human Genome
3 BILLION base pairs In each cell = 46 DNA strands
Mistakes can occur with mismatched pairs Called mutations
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DNA Repair
If no repairIn sex cells inherited diseasesExample Tay-Sachs disease - the body
lacks hexosaminidase A, a protein that helps break down a chemical found in nerve tissue
caused by a defective gene on chromosome 15
In somatic cells cancer
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Replication Mistakes
Change in the nucleotide base sequence of a genome; rare.
Almost always deleterious (bad). A deleterious mutation has a negative effect on the phenotype, and thus decreases the fitness of the organism. (A harmful mutation)
Rarely lead to a protein having a novel property that improves ability of organism and its descendents to survive and reproduce.
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Effect of Mutation
Sickle cell anemia is a disease passed down through families in which red blood cells form an abnormalsickle or crescent shape. Red blood cellscarry oxygen to thebody and are normallyshaped like a disc.Causes bone pain.