Replication of DNA

DNA Replication

Revised 10/31/2006

DNA Replication:

• Is a fundamental process

occurring in all living

organisms to copy their DNA.

Nature published Watson & Crick's manuscript describing the double helical structure of DNA, which marks the beginning of the modern era of genetics.

Their paper makes some implications about how DNA copies itself.

1953

The paper never comes right out and says how it happens (they didn’t really know for sure HOW it replicated at the time).

But they did suggest some properties of DNA that lend itself to being copied.

Nature of the Genetic Material

Property 1 - it must contain, in a stable form, information encoding the organism’s structure, function, development and reproduction

Property 2 - it must replicate accurately so progeny cells have the same genetic makeup

Property 3 - it must be capable of some variation (mutation) to permit evolution

So, an initial problem (whose solution was hinted at by Watson and Crick) was:

How is DNA replicated?

We need to review the research so far:

1928 - Griffith discovered a “transforming principle” in heat killed bacteria. (Supports Property 1)

1944 - Avery demonstrated that the transforming principle is sensitive to DNase.

1952 - Hershey & Chase used 32P and 35S labelling, of bacteriophage T2 DNA and protein respectively, to show only the DNA enters the host cell and can be passed to progeny phage. (Supports Property 2)

Meselson and Stahl (1958)

The exact method still had yet to be worked out. But, there were three similar but varying ideas about how this happened:

1. Conservative Model

2. Semiconservative Model

3. Dispersive Model.

Meselson and Stahl get the credit for working out which model was correct.

Their experiment is as follows:

Bacteria are grown in two solutions, one where the DNA they build must use N15 in their bases and one where it must use N14 in their bases. N15 has an extra neutron so it is heavier.

When the DNA is extracted from the N15 bacteria and centrifuged at high speed, it forms a band low in the test tube.

When the DNA is extracted from the N14 bacteria and centrifuged at high speed, it forms a band high in the test tube.

If bacteria were to be transferred from one media to the other, then have the DNA extracted from the bacteria and centrifuged at high speed, it should form 2 bands high in the test tube based on the fact that some of the DNA is made of N14 and N15 and some of it is made of only N14.

Possible Results in this test:

Meselson and Stahl: Grew the bacteria in N15 first, then

transferred some of it into N14 and centrifuged the DNA every 20 minutes.

This is what they found:

Proof for Semi-conservative model.

1958 Mathew Meselson and Frank Stahl

Replicated DNA:

Is passed to each new cell in each generation.

Creates complimentary strands.

The old strands serve as templates.

There are a few concepts that you need to understand before we get to a description of the actual process…

3’ (prime) vs 5’

If the #3 carbon on the deoxyribose molecule is NOT bonded to a phosphate below it, it is called 3’. Likewise for the #5 carbon at the top of the molecule.

• This a description of which carbon is at the end of the chain.

This is 5’

One backbone runs 3’ to 5’, the other runs 5’ to 3’.

• This arrangement is called Antiparallel.

This is going to be a problem during replication.

Replication of DNA involves unwinding the double helix and synthesizing two

new strands.

Nucleotides look a little different before

they are joined into the DNA molecule.

Remember that DNA is made up of

smaller subunits called Nucleotides.

They are called DNTPs before they are bonded together to form a DNA molecule.

• DNTP stands for Deoxynucleotide

Triphosphate.

• DNTP’s have 2 extra phosphates (compared

to nucleotides).

• As nucleotides are added to the growing DNA

backbone, the two extra phosphates break off

to provide energy to connect the nucleotide to

the rest of the chain.

Extra Phosphates Removed

Phosphodiester bond This is the name of the bond that links DNTPs into

the chain.

They are bonded into the chain via an oxygen in the phosphate.

To form a type of double-bond called an ester.

DNTP’s

NucleotidesALWAYSconnect to3’ carbon. So, DNA Synthesis occurs 5’ to 3’

Leading vs Lagging Strand

Leading Strand –is built as a single complete strand.

Lagging Strand –is built in segments that are connected later.

Replication Fork – The point where the 2 backbones are separated.

Replication Fork

Enzymes in Replication:•Helicase: Unwinds a portion of the DNA Double Helix.

•RNA Primase: Attaches RNA primers to the replicating

strands.

•DNA Polymerase III (delta): Binds to the 5' - 3' strand

in order to bring nucleotides and create the daughter

leading strand. Called Delta to distinguish it from the

enzyme on the other backbone.

•DNA Polymerase III (epsilon): Binds to the 3' - 5'

strand in order to create discontinuous segments

starting from different RNA primers. Called Epsilon to

distinguish it from the enzyme on the other backbone.

Enzymes in Replication:•RNase H: Removes RNA primers.

•Exonuclease (DNA Polymerase I): Finds areas

where RNA Primers were removed, and replaces

with DNA nucleotides.

•DNA Ligase: Adds phosphate in the remaining

gaps of the phosphate - sugar backbone.

•Nucleases: Remove wrong nucleotides from the

daughter strand.

Leading Strand:

DNA Helicase:• Unwinds and unzips the DNA double

helix.

Primase:• Adds RNA primers to DNA.

DNA Polymerase III:• Attaches dNTPs to the complimentary

bases in the template strand.

RNAse H:• Removes the RNA Primer.

DNA Polymerase I:• Adds DNA Nucleotides to replace

the RNA primers.

DNA Synthesis at the Origin

LEADING STRAND Replication occurs in

one continuous motion.

Lagging Strand:

Primase:• Attachment of RNA primers to

the DNA (RNA polymerase).

•Multiple sites of Primase attachment.

•This is because of the one-way synthesis of DNA

nucleotides on the three prime end.

DNA Polymerase III:

• AttachesDNA Nucleotides.

RNAse H:• Removes the RNA Primer.

DNA Polymerase I:

• Adds DNA Nucleotides.

Okazaki Fragments.

Okazaki

Fragments.

• LAGGING STRAND - Gluing together of the

Okazaki Fragments by the enzyme

Ligase.

Two

Strands

of DNA

Replication of DNA and Chromosomes

Speed of DNA replication:3,000 nucleotides/min in human 30,000 nucleotides/min in E.coli

Accuracy of DNA replication: Very precise. 1 error/1,000,000,000 nucleotides.

Replication Review Movie:

Where does replication start?

Different in Prokaryotes and eukaryotes.

Only One Replication Origin in E. coli

Bi-directional replication in E. coli

Multiple Origins in Eukaryotes

Eukaryotes have larger chromosomes than prokaryotes.

Multiple Origins in Eukaryotes

Replication speed 2,600 npm. Largest Drosophila (fruit fly) chromosome is

65,000,000 nucleotides long, but it can replicate in 3-4 min. – From a single origin, replication should take 8.5 days.– That means each chromosome must have some 7,000

origins of replication.

Replication Bubbles

Replication Bubbles

A replicating Drosophilachromosome

Origins initiate and replicate at different times.