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Science Biology DNA as

the gene c material DNA Molecular mechanism of DNA replica on
replica on
Roles of DNA polymerases and other replica on enzymes. Leading and lagging strands and
Speed and precision of Okazaki fragments.
DNA replica on

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Molecular structure of
DNA

Molecular mechanism of Key points:

DNA replica on
DNA replica on is semiconserva ve. Each strand in the double helix acts
as a template for synthesis of a new, complementary strand.
Mode of DNA replica on:
Meselson-Stahl
experiment
New DNA is made by enzymes called DNA polymerases, which require a
DNA proofreading and template and a primer (starter) and synthesize DNA in the 5' to 3'
repair direc on.

Telomeres and telomerase

During DNA replica on, one new strand (the leading strand) is made as a
Prac ce: DNA replica on
con nuous piece. The other (the lagging strand) is made in small pieces.

DNA replica on requires other enzymes in addi on to DNA polymerase,

including DNA primase, DNA helicase, DNA ligase, and topoisomerase.

Introduc on
DNA replica on, or the copying of a cell's DNA, is no simple task! There are
about 3 billion base pairs of DNA in your genome, all of which must be
accurately copied when any one of your trillions of cells divides1 .

The basic mechanisms of DNA replica on are similar across organisms. In this
ar cle, we'll focus on DNA replica on as it takes place in the bacterium E. coli,
but the mechanisms of replica on are similar in humans and other eukaryotes.

Let's take a look at the proteins and enzymes that carry out replica on, seeing
how they work together to ensure accurate and complete replica on of DNA.

The basic idea

DNA replica on is semiconserva ve, meaning that each strand in the DNA
double helix acts as a template for the synthesis of a new, complementary
strand.

This process takes us from one star ng molecule to two "daughter" molecules,
with each newly formed double helix containing one new and one old strand.

In a sense, that's all there is to DNA replica on! But what's actually most
interes ng about this process is how it's carried out in a cell.

Cells need to copy their DNA very quickly, and with very few errors (or risk
problem such as cancer). To do so, they use a variety of enzymes and proteins,
which work together to make sure DNA replica on is performed smoothly and
accurately.

DNA polymerase
One of the key molecules in DNA replica on is the enzyme DNA polymerase.
DNA polymerases are responsible for synthesizing DNA: they add nucleo des
one by one to the growing DNA chain, incorpora ng only those that are
complementary to the template.

Here are some key features of DNA polymerases:

They always need a template

They can only add nucleo des to the 3' end of a DNA strand

They can't start making a DNA chain from scratch, but require a pre-
exis ng chain or short stretch of nucleo des called a primer

They proofread, or check their work, removing the vast majority of

"wrong" nucleo des that are accidentally added to the chain

The addi on of nucleo des requires energy. This energy comes from the
nucleo des themselves, which have three phosphates a ached to them (much
like the energy-carrying molecule ATP). When the bond between phosphates
is broken, the energy released is used to form a bond between the incoming
nucleo de and the growing chain. [See the polymeriza on reac on]

In prokaryotes such as E. coli, there are two main DNA polymerases involved in
DNA replica on: DNA pol III (the major DNA-maker), and DNA pol I, which
plays a crucial suppor ng role we'll examine later.

Star ng DNA replica on

How do DNA polymerases and other replica on factors know where to begin?
Replica on always starts at specific loca ons on the DNA, which are called
origins of replica on and are recognized by their sequence.

E. coli, like most bacteria, has a single origin of replica on on its chromosome.
The origin is about 245 base pairs long and has mostly A/T base pairs (which
are held together by fewer hydrogen bonds than G/C base pairs), making the
DNA strands easier to separate.

Specialized proteins recognize the origin, bind to this site, and open up the
DNA. As the DNA opens, two Y-shaped structures called replica on forks are
formed, together making up what's called a replica on bubble. The replica on
forks will move in opposite direc ons as replica on proceeds.

Diagram based on similar illustra on in Reece et al. 2 .

How does replica on actually get going at the forks? Helicase is the first
replica on enzyme to load on at the origin of replica on3 . Helicase's job is to
move the replica on forks forward by "unwinding" the DNA (breaking the
hydrogen bonds between the nitrogenous base pairs).

Proteins called single-strand binding proteins coat the separated strands of

DNA near the replica on fork, keeping them from coming back together into a
double helix.

Primers and primase

DNA polymerases can only add nucleo des to the 3' end of an exis ng DNA
strand. (They use the free -OH group found at the 3' end as a "hook," adding a
nucleo de to this group in the polymeriza on reac on.) How, then, does DNA
polymerase add the first nucleo de at a new replica on fork?

Alone, it can't! The problem is solved with the help of an enzyme called
primase. Primase makes an RNA primer, or short stretch of nucleic acid
complementary to the template, that provides a 3' end for DNA polymerase to
work on. A typical primer is about five to ten nucleo des long. The primer
primes DNA synthesis, i.e., gets it started.

Once the RNA primer is in place, DNA polymerase "extends" it, adding
nucleo des one by one to make a new DNA strand that's complementary to
the template strand.

Leading and lagging strands

In E. coli, the DNA polymerase that handles most of the synthesis is DNA
polymerase III. There are two molecules of DNA polymerase III at a replica on
fork, each of them hard at work on one of the two new DNA strands.

DNA polymerases can only make DNA in the 5' to 3' direc on, and this poses
a problem during replica on. A DNA double helix is always an -parallel; in
other words, one strand runs in the 5' to 3' direc on, while the other runs in
the 3' to 5' direc on. This makes it necessary for the two new strands, which
are also an parallel to their templates, to be made in slightly different ways.

One new strand, which runs 5' to 3' towards the replica on fork, is the easy
one. This strand is made con nuously, because the DNA polymerase is moving
in the same direc on as the replica on fork. This con nuously synthesized
strand is called the leading strand.

The other new strand, which runs 5' to 3' away from the fork, is trickier. This
strand is made in fragments because, as the fork moves forward, the DNA
polymerase (which is moving away from the fork) must come off and rea ach
on the newly exposed DNA. This tricky strand, which is made in fragments, is
called the lagging strand.

The small fragments are called Okazaki fragments, named for the Japanese
scien st who discovered them. The leading strand can be extended from one
primer alone, whereas the lagging strand needs a new primer for each of the
short Okazaki fragments.

The maintenance and cleanup crew

Some other proteins and enzymes, in addi on the main ones above, are
needed to keep DNA replica on running smoothly. One is a protein called the
sliding clamp, which holds DNA polymerase III molecules in place as they
synthesize DNA. The sliding clamp is a ring-shaped protein and keeps the
DNA polymerase of the lagging strand from floa ng off when it re-starts at a
new Okazaki fragment4 .

Topoisomerase also plays an important maintenance role during DNA

replica on. This enzyme prevents the DNA double helix ahead of the
replica on fork from ge ng too ghtly wound as the DNA is opened up. It
acts by making temporary nicks in the helix to release the tension, then sealing
the nicks to avoid permanent damage.

Finally, there is a li le cleanup work to do if we want DNA that doesn't

contain any RNA or gaps. The RNA primers are removed and replaced by DNA
through the ac vity of DNA polymerase I, the other polymerase involved in
replica on. The nicks that remain a er the primers are replaced get sealed by
the enzyme DNA ligase.

Summary of DNA replica on in E. coli

Let's zoom out and see how the enzymes and proteins involved in replica on
work together to synthesize new DNA.

Helicase opens up the DNA at the replica on fork.

Single-strand binding proteins coat the DNA around the replica on fork
to prevent rewinding of the DNA.

Topoisomerase works at the region ahead of the replica on fork to

prevent supercoiling.

Primase synthesizes RNA primers complementary to the DNA strand.

DNA polymerase III extends the primers, adding on to the 3' end, to make
the bulk of the new DNA.

RNA primers are removed and replaced with DNA by DNA polymerase I.

The gaps between DNA fragments are sealed by DNA ligase.

DNA replica on in eukaryotes

The basics of DNA replica on are similar between bacteria and eukaryotes
such as humans, but there are also some differences:

Eukaryotes usually have mul ple linear chromosomes, each with mul ple
origins of replica on. Humans can have up to 100,000 origins of
replica on5 !

Most of the E. coli enzymes have counterparts in eukaryo c DNA

replica on, but a single enzyme in E. coli may be represented by mul ple
enzymes in eukaryotes. For instance, there are five human DNA
polymerases with important roles in replica on5 .

Most eukaryo c chromosomes are linear. Because of the way the lagging
strand is made, some DNA is lost from the ends of linear chromosomes
(the telomeres) in each round of replica on.

[References]

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Fairuz Nawar 3 years ago

Is topoisomerase same as DNA gyrase ?

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William Holbrook 3 years ago

DNA gyrase is a type of topoisomerase.

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Michelle Verstraaten 3 years ago

"Many DNA have proofreading ac vity" men ons : "In most cases, the correct
nucleo de is indeed added, because the DNA polymeriza on reac on won't
usually occur unless the incoming nucleo de base-pairs correctly with the
template." If the reac on cannot occur unless there is correct base matching, how
then can the DNA polymerase s ll make an error?

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emilyabrash 3 years ago

The key word is the "usually." The reac on won't occur with a mis-paired
base in most cases. However, about 1 in 10^5 base pairs will involve an
incorrect pairing. This may not same like a high rate of errors, but it is
high enough to cause a lot of muta ons in a cell. The role of the
proofreading is to fix these occasional but s ll problema c errors.

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natureforever.care 3 years ago

How are the histone proteins taken care of during eukaryo c DNA replica on?

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J 3 years ago

The DNA is first unwound at origins of replica on and the displaced

histone proteins move onto to other parts of the DNA that haven't been
unwound so that those parts can maintain their chroma n structure.

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Isaac D. Cohen a year ago

In the last sec on "DNA replica on in Eukaryotes" it says that in eukaryote cells a
li le DNA at the ends of the chromosomes gets lost. If this is the case, will we
eventually loose enough DNA to stop func oning properly?

You might say that this is indeed why we die eventually. Each me our cells divide
and our DNA gets copied some of it gets lost placing a limit on how many mes
our cells could divide and s ll func on properly. However, consider that DNA is
also copied before meiosis. This means that the DNA that was lost when the
ancestors of my cells (in my parents) divided was never passed on to me. And the
DNA that got lost when my cells divided to form my germ cells will never get
passed on to my sperm cells. Will humanity eventually loose its en re gene pool?

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Charles LaCour a year ago

At the ends of DNA strands there is a sec on non-coding nucleo des

that we call a telomere. The telomere is what gets shorter every me a
cell divides and when the telomere is gone is when the cell
spontaneously dies. There is no loss of coding DNA in this process so
there is no loss of gene c informa on between genera ons.

2 comments (6 votes) Upvote Downvote Flag more

Lilah Bleich 3 years ago

Why are the DNA polymerases numbered here? (I/II/III) I though that Eu-k were
named by alpha beta delta etc

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Daltara Darana 3 years ago

Prokaryotes have DNA polymerases I, II, III, eukaryotes have alpha, delta,
epsilon and such.

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grega ac 3 years ago

The part of the ar cle that deals with the Okazaki-fragments states that:

"DNA polymerase I and DNA ligase are also needed (more infrequently) for the
leading strand. DNA polymerase I removes the primer at the very beginning of the
strand, and DNA polymerase seals the remaining gap."

Shouldn't the gap between the Primerreplacement and the new Nucleo de chain
be sealed by DNA-Ligase instead?

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emilyabrash 3 years ago

Yep, that was a typo! I've fixed it now and it should be corrected on the
site shortly. Thanks for no cing!

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Hans Kris an Pedersen 3 years ago

In the paragraph 'DNA polymerases' it says that polymerase II has a DNA repair
func on, but in 'Many DNA polymerases have proofreading ac vity', it is stated
that DNA pol. I and II have proofreading ac vity. Does DNA pol. II aid in a different
repair mechanism than proofreading?

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emilyabrash 3 years ago

Great ques on! Yes, DNA polymerase II is involved in repair of damage

that occurs outside the context of DNA replica on, such as cross-links
between strands caused by certain chemical agents. There is a li le more
detail in the Wikipedia ar cle if you are curious:
h ps://en.wikipedia.org/wiki/DNA_polymerase_II.

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caramel.priyanka 6 months ago

Genome refers to the haploid content of DNA in a cell, so how can it consist of 3
billion base PAIRS? Or is it the diploid content in any cell?

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tyersome 6 months ago

Most DNA exists as a double-stranded DNA in a double helix — the

strands are held together by base pairs and we usually think of this as a
single molecule (even though there are no covalent bonds between the
two strands).

So, each haploid chromosome has at its core a (mostly) double-stranded

DNA "molecule" and a human haploid genome contains ~3.2 billion base
pairs.

A diploid cell has two of each haploid chromosome (each of which

contains a double-stranded DNA "molecule"), so a diploid human genome
contains ~6.4 billion base pairs.

Does that help?

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Shreyas Pai 3 years ago

'A DNA molecule “unzips” as the hydrogen bonds between bases are broken,
separa ng the two strands.' What makes this happen?

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krabas 3 years ago

Helicase enzyme. It binds at replica on ini a on site and moves along

DNA, in front of polymerase III, opening replica on fork.

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caramel.priyanka 6 months ago

How does topoisomerase prevent supercoiling of the DNA strands?

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tyersome 6 months ago

Good ques on!

Type 1§ topoisomerases act by making a "nick" in the DNA (cu ng one

strand of the double helix) — this allows the DNA to be unwound since
there is now a place where only one single-bond is holding the two parts
of the DNA molecule together. (Remember that single covalent bonds
can freely rotate.)

A er the supercoiling has been "unwound" these topoisomerases then

ligate the nicked strand back together recrea ng an unbroken double-
stranded DNA double-helix.

This is also discussed on this website:

h ps://www.ebi.ac.uk/interpro/potm/2006_1/Page1.htm

§There are also type 2 topoisomerases (o en known as gyrases) that cut

both strands.

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Molecular structure of DNA Mode of DNA replica on: Meselson-Stahl experiment

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