DNA Replication

Biology 12

•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.

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)

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

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

6

Chargraff’s Rule:Chargraff’s Rule:

• Adenine and Thymine always join together

A T

• Cytosine and Guanine always join together

C G

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

PO4

PO4

PO4

thymine

PO4

PO4

PO4

PO4

adenine

cytosine

PO4

guanine

Bonding 2 11

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

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.

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?

DNA is very long. VERY long. How can a very long DNA molecule get itself replicated in a relatively short period of time?

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?

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?

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)?

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.

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

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

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)

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”

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.

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.

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.

- 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

Separating DNA StrandsSSBs (Single-stranded binding proteins)

Bind to the exposed DNAKeep from “annealing”

• Reattaching with complimentary base pair

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.

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.

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.

- 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.

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.

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’

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’

Figure 5

III

Parent strand

Replication fork

Single-stranded binding proteins

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.

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’

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

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’

DNA ligase Joins Okazaki fragments together

Completes backboneLagging strand only!

Building Complimentary Strands

Leading strand Built continuously From parent 3’ end toward replication

fork

Building Complimentary Strands

Lagging strand Built in short Okazaki fragments

between RNA primersDiscontinuousFrom parent 3’ end away from

replication fork

III

Parent strand

Replication fork

Single-stranded binding proteins

Okazaki fragments

Step 1

Step 2

Step 3

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.

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

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.

Proofreading

DNA polymerase I and III Check for proper base

pairing If mistake found:

Removes Replace with proper

nucleotide

- Results in two identical daughter DNA strandsResults in two identical daughter DNA strands

- ½ new and ½ old½ new and ½ old

End Result

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)

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

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

Human Genome

3 BILLION base pairs In each cell = 46 DNA strands

Mistakes can occur with mismatched pairs Called mutations

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

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.

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.