Explain the steps involved in DNA replication.

During DNA replication, two molecules of DNA are made from one. When a DNA molecule is

copied, each new molecule contains one strand of parental DNA and one stand of new DNA (See

figure 17.19 on page 582 in your text).

There are 4 main stages:

1. Initiation: When a portion of the double helix is unwound.

2. Elongation: When two new strands of DNA are assembled.

3. Termination: When the new DNA molecules re-form into helixes.

4. Proofreading and Correction: Occurs throughout the process to minimize the errors

that may occur.

Step 1: Initiation: Unwinding of the double helix:

» The process of replication begins in the DNA molecules at thousands of sites called origins of

replication. At these sites, which look like little bubbles, the hydrogen bonds between the bases

are broken and the paired bases separate. After a replication bubble has been opened, molecules

of an enzyme called DNA polymerase insert themselves into the space between the two strands.

The helix begins to pull apart or unwind.

» The unwinding of the helix is facilitated by an enzyme called helicase, which is part of the

replication complex - a group of enzymes and other proteins that take care of the replication

process. There are two replication complexes at each origin of replication. As unwinding

continues, they move in opposite directions creating two Y-shaped replication forks.

Replication proceeds in both directions until the bubbles meet.

The image below shows an origin of replication. The green arrows indicate the directions that the

helix will unravel. New bases (in blue) are coming in and attaching to their complementary bases

on the old strand.

You will find this diagram on page 583 in your text.

Step 2 Elongation: Assembly of 2 new strands of DNA:

● DNA polymerase (which is the enzyme that inserts itself into the space between the two

strands) attaches new nucleotides to the free 3’ hydroxyl end. This imposes two conditions on

the elongation process:

- First, replication can only take place in the 5’ and 3’ direction (leading strand).

and

- second, a short strand or RNA known as a primer must be available to serve as the

starting point for the attachment of new nucleotides. The primer simply gets the bases primed to

receive new bases that will form the new DNA strand.

● During replication, much of the newly formed DNA is found in short fragments of one to two

thousand nucleotides in prokaryotes and a few hundred nucleotides in eukaryotes. These

fragments are known as Okazaki Fragments. These fragments occur during the elongation of

the daughter DNA strand that must be built in the 3’ to 5’ direction (lagging strand).

● There are 2 strands during the replication process. They are:

- Leading Strand: This is the strand in which the elongation process continuously proceeds in

the 5’ and 3’ direction. The elongation proceeds in the same direction as the movement of the

replication fork.

- Lagging Strand: This strand is manufactured more slowly than the leading strand.

DNA polymerase adds nucleotides in fragments called Okazaki Fragments which are

eventually spliced together by the enzyme DNA ligase. Replication of this strand takes place in

the opposite direction to the movement of the replication fork.

● Another enzyme called primase synthesizes an RNA primer to begin the elongation process.

Only one primer is needed on the leading strand. A new primer is needed for each Okazaki

Fragment on the lagging strand.

Step Three: Termination:

● Once the new strands are complete, the daughter DNA molecules rewind automatically back

to their original helix structure.

● The problem with the end of a linear chromosome with the RNA primer has been removed

from the 5’ end of each daughter strand, there is no adjacent fragment onto which new DNA

nucleotides can be added to fill the gap. The result is that each replication results in slightly

shorter daughter chromosomes.

● Eukaryotes have special buffer zones called telomeres at the end of each chromosome to

guard against this problem. These are highly repetitive nucleotide sequences typically rich in G

nucleotides. These regions do not direct cell development.

Step Four: Proofreading and Correction:

● This step of DNA replication ensures accuracy of replication.

● After each new nucleotide is added to a new DNA strand, DNA polymerase can recognize

whether or not hydrogen bonding is taking place between base pairs.

● Absence of hydrogen bonding indicates a mismatch. DNA polymerase excises the incorrect

base and then adds the correct nucleotide.

● This double check increases accuracy to a factor of about one error per billion base pairs.

Key Enzymes in DNA replication:

ENZYME GROUP

Helicase

DNA Polymerase

DNA Ligase

Primase

FUNCTION

Cleaves and unwinds short sections of

DNA ahead of the replication fork.

Serves 3 different functions:

1. Adds new nucleotides to 3’ end

of elongating strand.

2. Dismantles RNA primer.

3. Proofreads base pairings

Catalyzes the formation of phosphate

bridges between nucleotides to join

Okazaki Fragments.

Synthesizes an RNA primer to begin the

elongation process.

http://www.chemguide.co.uk/organicprops/aminoacids/dna2.html

Semi-conservative replication

A very simple look at the process

We'll explain exactly what "semi-conservative" means when we have got some diagrams to look at. First imagine what happens if the two individual strands in the DNA double helix start to unzip.

The diagram shows this happening in the middle of the DNA double helix - you mustn't assume that the top of the diagram is the end of the chain. It isn't. Further up the double helix, the two strands will still be joined together.

In fact, this is happening lots of times along the very long DNA molecule. Lengths of chain become separated to form what are known as "bubbles". If you feel the need to see this in more detail, read the rest of this page, and then have a quick look at the links

Some of the hydrogen bonds get broken and the two strands become partly separated.

The red dotted lines on the diagram just point out the original base pairs. These are not bonds in

any form. These base pairs are now much too far apart for any sort of bonding between them.

Now suppose that you have a source of nucleotides - phosphate joined to deoxyribose joined to a

base, including all the four sorts of bases needed for DNA.

The next diagram shows what would happen if a nucleotide containing guanine (G) and one

containing cytosine (C) were attracted to the top two bases on the left-hand strand of the

unzipped DNA - and then joined together.

How did they end up joined together? This is all under the control of a number of enzymes, one

of which (DNA polymerase) is responsible for joining up nucleotides along the chain in this way.

Now suppose the same sort of thing happened at the top of the right-hand strand. You would end

up with . . .

Now compare the double strands that you are forming on the left- and right-hand sides. They are

exactly the same . . . and if you were to continue this process, they would continue to be the

same.

And if you compare the patterns of bases in the new DNA being formed with what was in the

original DNA before it started to unzip, everything is the same. This is inevitable because of the

way the bases pair together.

What does semi-conservative replication mean?

Let's simplify the last diagram, and assume that the whole copying process is complete. The next

diagram focusses on the short bit of the total DNA molecule that we have been looking at. A

typical human DNA molecule is around 150 million base pairs long - you will have to imagine

the rest of it!

You have also got to remember that in reality the whole thing would have coiled into its double

helix. Trying to draw that just makes everything look messy and complicated!

The original DNA is shown all in blue. The red strands in the daughter DNA are the ones which

have been built on the original blue strands during the replication process.

You can see that each of the daughter molecules is made of half of the original DNA plus a new

strand. That's all "semi-conservative replication" means. Half of the original DNA is conserved

(kept) in each of the daughter molecules.

The red and blue, of course, have no physical significance apart from as a way of making the

diagrams clearer. All three of these DNA molecules will be identical in every way.

Below is the link you viewed during the smart-board presentation on DNA

replication. Hopefully you can view this link. It is a great resource for

visualizing and understanding the replication process.

http://www.johnkyrk.com/DNAreplication.html