DNA

REPLICATION
Presented by:
RAFAEL PINEDA ALEMAN
Molecular mechanism of DNA
replication
 DNA replication is semiconservative. Each strand in the double
helix acts as a template for synthesis of a new, complementary
strand.

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 New DNA is made by enzymes called DNA polymerases, which
require a template and a primer (starter) and synthesize DNA in
the 5' to 3' direction

 During DNA replication, one new strand (the leading strand) is

made as a continuous piece. The other (the lagging strand) is
made in small pieces.

 DNA replication requires other enzymes in addition to DNA

polymerase, including DNA primase, DNA helicase, DNA ligase,
and topoisomerase. 2
DNA Replication
DNA replication, 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

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cells divides

The basic mechanisms of DNA replication are

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

Let's take a look at the proteins and enzymes that

carry out replication, seeing how they work together
to ensure accurate and complete replication of DNA
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DNA Replication
The basic idea

DNA replication is semiconservative, meaning that

each strand in the DNA double helix acts as a
template for the synthesis of a new, complementary

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strand.

This process takes us from one starting molecule to

two "daughter" molecules, with each newly formed
double helix containing one new and one old strand

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DNA Replication
The basic idea

In a sense, that's all there is to DNA replication! But

what's actually most interesting about this process is

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how it's carried out in a cell.

Cells need to copy their DNA very quickly, and with

very few errors (or risk problems such as cancer). To
do so, they use a variety of enzymes and proteins,
which work together to make sure DNA replication is
performed smoothly and accurately

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DNA Replication
DNA polymerase

One of the key molecules in DNA replication is

the enzyme DNA polymerase. DNA
polymerases are responsible for synthesizing

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DNA: they add nucleotides one by one to the
growing DNA chain, incorporating only those
that are complementary to the template.

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DNA Replication
DNA polymerase

Here are some key features of DNA polymerases:

They always need a template

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

They can't start making a DNA chain from scratch, but

require a pre-existing chain or short stretch of
nucleotides called a primer

They proofread, or check their work, removing the vast

majority of "wrong" nucleotides that are accidentally
added to the chain

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DNA Replication
DNA polymerase

The addition of nucleotides requires energy. This

energy comes from the nucleotides themselves,
which have three phosphates attached to them

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(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 nucleotide and the growing chain

In prokaryotes such as E. coli, there are two main

DNA polymerases involved in DNA replication:
DNA pol III (the major DNA-maker), and DNA pol
I, which plays a crucial supporting role we'll
examine later
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DNA Replication
Starting DNA replication

How do DNA polymerases and other replication factors know

where to begin? Replication always starts at specific locations
on the DNA, which are called origins of replication and are

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recognized by their sequence.

E. coli, like most bacteria, has a single origin of replication

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
replication forks are formed, together making up what's called a
replication bubble. The replication forks will move in opposite
directions as replication proceeds 9
DNA Replication
Starting DNA replication

How does replication actually get going at the forks?

Helicase is the first replication enzyme to load on at
the origin of replication. Helicase's job is to move the

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replication 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 replication fork,
keeping them from coming back together into a double
helix

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DNA Replication
Primers and primase

DNA polymerases can only add nucleotides to the 3' end of an

existing DNA strand. (They use the free -OH group found at the 3'
end as a "hook," adding a nucleotide to this group in the

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polymerization reaction.) How, then, does DNA polymerase add the
first nucleotide at a new replication 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
nucleotides long. The primer primes DNA synthesis, i.e., gets it
started.

Once the RNA primer is in place, DNA polymerase "extends" it,

adding nucleotides one by one to make a new DNA strand that's
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complementary to the template strand.
DNA Replication
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 replication fork, each of them hard at work on one of the two

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new DNA strands.

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

One new strand, which runs 5' to 3' towards the replication fork, is
the easy one. This strand is made continuously, because the DNA
polymerase is moving in the same direction as the replication fork.
This continuously synthesized strand is called the leading strand 12
DNA Replication
Leading and lagging strands

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

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polymerase (which is moving away from the fork)
must come off and reattach 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 scientist 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

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DNA Replication
The maintenance and cleanup crew

Some other proteins and enzymes, in addition the main

ones above, are needed to keep DNA replication running
smoothly. One is a protein called the sliding clamp, which

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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 floating off when it re-starts at a new Okazaki
fragment^4

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DNA Replication
The maintenance and cleanup crew

Topoisomerase also plays an important maintenance role during

DNA replication. This enzyme prevents the DNA double helix ahead
of the replication fork from getting too tightly wound as the DNA is

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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 little 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 activity of DNA polymerase I, the
other polymerase involved in replication. The nicks that remain after
the primers are replaced get sealed by the enzyme DNA ligase.

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DNA Replication
Helicase opens up the DNA at the replication fork.

Single-strand binding proteins coat the DNA around

the replication fork to prevent rewinding of the DNA.

Topoisomerase works at the region ahead of the

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replication 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 16

ligase
DNA Replication

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DNA Replication
DNA replication in eukaryotes

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

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 Eukaryotes usually have multiple linear chromosomes, each with multiple
origins of replication. Humans can have up to 100,000 origins of
replication!

 Most of the E. coli enzymes have counterparts in eukaryotic DNA

replication, but a single enzyme in E. coli may be represented by multiple
enzymes in eukaryotes. For instance, there are five human DNA
polymerases with important roles in replication.

 Most eukaryotic 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 replication 18
Overview of transcription
Key points:

 Transcription is the first step in gene expression. It involves

copying a gene's DNA sequence to make an RNA molecule.

 Transcription is performed by enzymes called RNA polymerases,

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which link nucleotides to form an RNA strand (using a DNA
strand as a template).

 Transcription has three stages: initiation, elongation, and

termination.
 In eukaryotes, RNA molecules must be processed after
transcription: they are spliced and have a 5' cap and poly-A tail
put on their ends.

 Transcription is controlled separately for each gene in your

genome.
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Overview of transcription
Have you ever had to transcribe something? Maybe someone
left a message on your voicemail, and you had to write it
down on paper. Or maybe you took notes in class, then
rewrote them neatly to help you review.

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As these examples show, transcription is a process in which
information is rewritten. Transcription is something we do in
our everyday lives, and it's also something our cells must do,
in a more specialized and narrowly defined way. In biology,
transcription is the process of copying out the DNA sequence
of a gene in the similar alphabet of RNA.

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Overview of transcription
Transcription is the first step in gene
expression, in which information from a gene
is used to construct a functional product such
as a protein. The goal of transcription is to

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make a RNA copy of a gene's DNA sequence.
For a protein-coding gene, the RNA copy, or
transcript, carries the information needed to
build a polypeptide (protein or protein
subunit). Eukaryotic transcripts need to go
through some processing steps before
translation into proteins.

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Overview of transcription
RNA polymerase

The main enzyme involved in transcription is RNA

polymerase, which uses a single-stranded DNA
template to synthesize a complementary strand of RNA.

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Specifically, RNA polymerase builds an RNA strand in
the 5' to 3' direction, adding each new nucleotide to the
3' end of the strand.

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Overview of transcription
Stages of transcription

Transcription of a gene takes place in three stages:

initiation, elongation, and termination. Here, we will
briefly see how these steps happen in bacteria. You can

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learn more about the details of each stage (and about
how eukaryotic transcription is different) in the stages
of transcription article.

Initiation. RNA polymerase binds to a sequence of

DNA called the promoter, found near the beginning of a
gene. Each gene (or group of co-transcribed genes, in
bacteria) has its own promoter. Once bound, RNA
polymerase separates the DNA strands, providing the
single-stranded template needed for transcription.

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Overview of transcription
Stages of transcription

Elongation. One strand of DNA, the template

strand, acts as a template for RNA polymerase. As

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it "reads" this template one base at a time, the
polymerase builds an RNA molecule out of
complementary nucleotides, making a chain that
grows from 5' to 3'. The RNA transcript carries the
same information as the non-template (coding)
strand of DNA, but it contains the base uracil (U)
instead of thymine (T). [What do 5' and 3' mean?]

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Overview of transcription
Stages of transcription

Termination. Sequences called terminators signal that the RNA

transcript is complete. Once they are transcribed, they cause the
transcript to be released from the RNA polymerase. An example of

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a termination mechanism involving formation of a hairpin in the
RNA is shown below

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Overview of transcription
Eukaryotic RNA modifications

In bacteria, RNA transcripts can act as

messenger RNAs (mRNAs) right away.
In eukaryotes, the transcript of a protein-
coding gene is called a pre-mRNA and

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must go through extra processing before
it can direct translation.

 Eukaryotic pre-mRNAs must have their ends

modified, by addition of a 5' cap (at the beginning)
and 3' poly-A tail (at the end).

 Many eukaryotic pre-mRNAs undergo splicing. In

this process, parts of the pre-mRNA (called
introns) are chopped out, and the remaining pieces
(called exons) are stuck back together. 26
Overview of transcription
Eukaryotic RNA modifications

 End modifications increase the

stability of the mRNA, while splicing

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gives the mRNA its correct sequence.
(If the introns are not removed, they'll
be translated along with the exons,
producing a "gibberish" polypeptide.)

 To learn more about pre-mRNA

modifications in eukaryotes, check out
the article on pre-mRNA processing.

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Overview of transcription
Transcription happens for individual genes

Not all genes are transcribed all the time. Instead,

transcription is controlled individually for each gene
(or, in bacteria, for small groups of genes that are

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transcribed together). Cells carefully regulate
transcription, transcribing just the genes whose
products are needed at a particular moment.

For example, the diagram below shows a "snapshot"

of an imaginary cell's RNAs at a given moment in
time. In this cell, genes 1, 2 and 3, are transcribed,
while gene 4 is not. Also, genes 1, 2, and 3 are
transcribed at different levels, meaning that different
numbers of RNA molecules are made for each

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Eukaryotic pre-mRNA processing
Key points:

 When an RNA transcript is first made in a eukaryotic cell, it is

considered a pre-mRNA and must be processed into a messenger
RNA (mRNA).

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 A 5' cap is added to the beginning of the RNA transcript, and a 3'
poly-A tail is added to the end.

 In splicing, some sections of the RNA transcript (introns) are

removed, and the remaining sections (exons) are stuck back
together.

 Some genes can be alternatively spliced, leading to the

production of different mature mRNA molecules from the same
initial transcript.
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Eukaryotic pre-mRNA processing
gene expression (the "reading out" of a gene to make a protein, or
chunk of a protein) happens a little bit differently in bacteria and
eukaryotes such as humans.
In bacteria, RNA transcripts are
ready to act as messenger RNAs

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and get translated into proteins
right away. In eukaryotes,
things are a little more complex,
though in an pretty interesting
way

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Eukaryotic pre-mRNA processing
he molecule that's directly made by transcription
in one of your (eukaryotic) cells is called a pre-
mRNA, reflecting that it needs to go through a
few more steps to become an actual messenger
RNA (mRNA). These are:

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Addition of a 5' cap to the beginning of the RNA

Addition of a poly-A tail (tail of A nucleotides)

to the end of the RNA

Chopping out of introns, or "junk" sequences,

and pasting together of the remaining, good
sequences (exons)

Once it's completed these steps, the RNA is a mature mRNA. It can
travel out of the nucleus and be used to make a protein. 31
Eukaryotic pre-mRNA processing
Both ends of a pre-mRNA are modified by the addition of chemical
groups. The group at the beginning (5' end) is called a cap, while the
group at the end (3' end) is called a tail. Both the cap and the tail
protect the transcript and help it get exported from the nucleus and
translated on the ribosomes (protein-making "machines") found in

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the cytosol.

The 5’ cap is added to the first nucleotide in the transcript during

transcription. The cap is a modified guanine (G) nucleotide, and it
protects the transcript from being broken down. It also helps the
ribosome attach to the mRNA and start reading it to make a protein

32
Eukaryotic pre-mRNA processing
How is the poly-A tail added? The 3' end of the RNA forms in kind
of a bizarre way. When a sequence called a polyadenylation signal
shows up in an RNA molecule during transcription, an enzyme
chops the RNA in two at that site. Another enzyme adds about 100 –
200 adenine (A) nucleotides to the cut end, forming a poly-A tail.

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The tail makes the transcript more stable and helps it get exported
from the nucleus to the cytosol.

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Eukaryotic pre-mRNA processing
RNA splicing

The third big RNA processing event that happens in your cells is
RNA splicing. In RNA splicing, specific parts of the pre-mRNA,
called introns are recognized and removed by a protein-and-RNA

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complex called the spliceosome. Introns can be viewed as "junk"
sequences that must be cut out so the "good parts version" of the
RNA molecule can be assembled.

What are the "good parts"? The pieces of the RNA that are not
chopped out are called exons. The exons are pasted together by the
spliceosome to make the final, mature mRNA that is shipped out of
the nucleus

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Eukaryotic pre-mRNA processing
RNA splicing

A key point here is that it's only the exons of a gene that encode a protein. Not only do the introns not
carry information to build a protein, they actually have to be removed in order for the mRNA to encode a
protein with the right sequence. If the spliceosome fails to remove an intron, an mRNA with extra "junk"

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in it will be made, and a wrong protein will get produced during translation

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Eukaryotic pre-mRNA processing
Alternative splicing

Why splice? We don't know for sure

why splicing exists, and in some ways,
it seems like a wasteful system.

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However, splicing does allow for a
process called alternative splicing, in
which more than one mRNA can be
made from the same gene. Through
alternative splicing, we (and other
eukaryotes) can sneakily encode more
different proteins than we have genes in
our DNA

36
Eukaryotic pre-mRNA processing
Alternative splicing

In alternative splicing, one pre-mRNA may

be spliced in either of two (or sometimes

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many more than two!) different ways. For
example, in the diagram below, the same
pre-mRNA can be spliced in three different
ways, depending on which exons are kept.
This results in three different mature
mRNAs, each of which translates into a
protein with a different structure.

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Overview of translation
Take a moment to look at your hands. The bone,
skin, and muscle you see are made up of cells.
And each of those cells contains many millions of
proteins. As a matter of fact, proteins are key
molecular "building blocks" for every organism
on Earth!
How are these proteins made in a cell? For
starters, the instructions for making proteins are
"written" in a cell’s DNA in the form of genes
Basically, a gene is used to build a protein in a two-step
process:
Step 1: transcription! Here, the DNA sequence of a gene is
"rewritten" in the form of RNA. In eukaryotes like you and
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me, the RNA is processed (and often has a few bits snipped
out of it) to make the final product, called a messenger
RNA or mRNA.
Step 2: translation! In this stage, the mRNA is "decoded"
to build a protein (or a chunk/subunit of a protein) that
contains a specific series of amino acids. [What exactly is
an "amino acid"?]
38
The genetic code
During translation, a cell “reads” the
information in a messenger RNA (mRNA)
and uses it to build a protein. Actually, to be a
little more techical, an mRNA doesn’t always
encode provide instructions for a whole

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protein. Instead, what we can confidently say
is that it always encodes a polypeptide, or
chain of amino acids.

In an mRNA, the instructions for building a

polypeptide are RNA nucleotides (As, Us, Cs,
and Gs) read in groups of three. These groups
of three are called codons.

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The genetic code
There are 61 codons for amino acids, and each of
them is "read" to specify a certain amino acid out of
the 20 commonly found in proteins. One codon,
AUG, specifies the amino acid methionine and also
acts as a start codon to signal the start of protein

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construction.

There are three more codons that do not specify

amino acids. These stop codons, UAA, UAG, and
UGA, tell the cell when a polypeptide is complete.
All together, this collection of codon-amino acid
relationships is called the genetic code, because it lets
cells “decode” an mRNA into a chain of amino acids.

40
Overview of translation
How is an mRNA "read" to make a polypeptide? Two
types of molecules with key roles in translation are
tRNAs and ribosomes.

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Transfer RNAs (tRNAs)

Transfer RNAs, or tRNAs, are molecular "bridges" that

connect mRNA codons to the amino acids they encode.
One end of each tRNA has a sequence of three
nucleotides called an anticodon, which can bind to
specific mRNA codons. The other end of the tRNA
carries the amino acid specified by the codons.

There are many different types of tRNAs. Each type

reads one or a few codons and brings the right amino
acid matching those codons.
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Ribosomes
Ribosomes are the structures where polypeptides
(proteins) are built. They are made up of protein
and RNA (ribosomal RNA, or rRNA). Each
ribosome has two subunits, a large one and a small
one, which come together around an mRNA—kind

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of like the two halves of a hamburger bun coming
together around the patty.

The ribosome provides a set of handy slots where

tRNAs can find their matching codons on the
mRNA template and deliver their amino acids.
These slots are called the A, P, and E sites. Not
only that, but the ribosome also acts as an enzyme,
catalyzing the chemical reaction that links amino
acids together to make a chain.

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Steps of translation
Your cells are making new proteins every second of
the day. And each of those proteins must contain the
right set of amino acids, linked together in just the
right order. That may sound like a challenging task,
but luckily, your cells (along with those of other

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animals, plants, and bacteria) are up to the job.

To see how cells make proteins, let's divide

translation into three stages: initiation (starting off),
elongation (adding on to the protein chain), and
termination (finishing up).

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Steps of translation
Getting started: Initiation

In initiation, the ribosome assembles around the

mRNA to be read and the first tRNA (carrying the
amino acid methionine, which matches the start

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codon, AUG). This setup, called the initiation
complex, is needed in order for translation to get
started.

Extending the chain: Elongation

Elongation is the stage where the amino acid chain

gets longer. In elongation, the mRNA is read one
codon at a time, and the amino acid matching each
codon is added to a growing protein chain.

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Steps of translation
Each time a new codon is exposed:

 A matching tRNA binds to the codon

 The existing amino acid chain (polypeptide)
is linked onto the amino acid of the tRNA

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via a chemical reaction
 The mRNA is shifted one codon over in the
ribosome, exposing a new codon for reading

During elongation, tRNAs move through the A,

P, and E sites of the ribosome, as shown above.
This process repeats many times as new codons
are read and new amino acids are added to the
chain.

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Overview of translation
Finishing up: Termination

Termination is the stage in which the finished

polypeptide chain is released. It begins when a stop
codon (UAG, UAA, or UGA) enters the ribosome,

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triggering a series of events that separate the chain
from its tRNA and allow it to drift out of the
ribosome.

After termination, the polypeptide may still need to

fold into the right 3D shape, undergo processing
(such as the removal of amino acids), get shipped to
the right place in the cell, or combine with other
polypeptides before it can do its job as a functional
protein.

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 Grupo 5: Grupo 2: Grupo 6:
Grupo 1:
Tema: Generalidades Tema: Uso de la Tema: Uso de la
Tema: Generalidades
de Biotecnologia Biotecnologia Biotecnologia
de Biotecnologia
 Laura Muñoz  Jaime Jhonson,  Gissel Martinez
 Pamela Barrios
 Gabriela Gomez  Juan Martinez  Juan Alejandro Ruiz
 Yzze Howard
 Sara Restrepo  Samuel Laverde  Laura Rivas
 Camila Loaiza
 Maria Jose Gomez  Luis Elles  Niger Rodriguez
 Helen Machado
 Jukiana Cordoba  Felipe Arboleda
 Lauren Cuadrado
 Maria Alejandra  Alejandro Vergara

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 Danna Revollo
Marquez

Grupo 3: Grupo 4:
Tema: Tecnicas Tema: Transgenicos Temas:
Biotecnologicas  Alejandra Corredor Generalidades de Biotecnologia
 Melissa Ferreira  Ana Paternina, Uso de la Biotecnologia
 Ángel Velasco  Isabella Garcia Tecnicas Biotecnologicas
 José Rivas  Gabriella Gaviria, Transgenicos
 Melanie López  Marinella Cera
 Andrés Martínez  Laudith Barrante Taller traduccion
47
 https://www.khanacademy.org/science/ap-
biology/gene-expression-and-
regulation/transcription-and-rna-
processing/a/overview-of-transcription

https://www.khanacademy.org/science/ap-
biology/gene-expression-and-

Gracias
regulation/translation/a/translation-overview