DNA DNA REPLICATIONREPLICATION

Revising DNA structureRevising DNA structure

Why replicate?Why replicate?

Semiconservative replicationSemiconservative replication

Replication - overviewReplication - overview

Leading strandLeading strand

Lagging strandLagging strand

Central Dogma of Central Dogma of Information FlowInformation Flow

What is DNA replication?What is DNA replication?

DNA replicationDNA replication is the synthesis of DNA is the synthesis of DNA using itself as a template.using itself as a template.

Three stages: Three stages:

1.1. Initiation Initiation

2.2. Elongation Elongation

3.3. TerminationTermination

DNA NucleotideDNA Nucleotide

OO=P-O O

PhosphatePhosphate GroupGroup

CH2

O

CC

C C

SugarSugar(deoxyribose)(deoxyribose)

NNitrogenous baseNitrogenous base (A, G, C, or T)(A, G, C, or T)

DNA Double HelixDNA Double Helix

P

P

P

O

O

O

1

23

4

5

5

3

3

5

P

P

PO

O

O

1

2 3

4

5

5

3

5

3

G C

T A

When does DNA replication When does DNA replication take place?take place?

Every time cells need to duplicate Every time cells need to duplicate themselves: themselves:

During growthDuring growth During embryo development. During embryo development. To repair damaged tissues, e.g. after an To repair damaged tissues, e.g. after an

injury or wound.injury or wound. To replace damaged or dead cells.To replace damaged or dead cells. DNA replication takes places only once DNA replication takes places only once

in a cell’s cycle, BEFORE cell division.in a cell’s cycle, BEFORE cell division.

Complementary base pairing allows each strand of DNA to serve as a template for DNA replication.

After replication is completed, two identical DNA molecules are obtained. DNA is a perfect illustration of function following form (structure dictates function).

Something old and Something old and something newsomething new

During DNA replication, both strands are “copied”, that is, each one acts as a model to

make a new strand.

This type of replication is known as

SEMICONSERVATIVESEMICONSERVATIVE, since in each DNA molecule there is always an old strand, the

original template and a new strand recently made.

DNA replication is DNA replication is semiconservativesemiconservative

ReplicationReplication IIReplication can be Replication can be

summarised in 3 summarised in 3 steps:steps:

1)1) Separate the original Separate the original strandsstrands

2)2) Use each strand as a Use each strand as a template and make a template and make a new new complementary complementary strandstrand using free using free nucleotides.nucleotides.

3)3) Join the nucleotides in Join the nucleotides in each strand to form a each strand to form a chain.chain.

As a result, 2 identical As a result, 2 identical DNA molecules are DNA molecules are obtained. obtained.

Replication II: the Replication II: the playersplayers

DNA replication is a very complex process DNA replication is a very complex process involving several proteins and enzymes. In involving several proteins and enzymes. In bacteria, these are:bacteria, these are:

The helicaseThe helicase The topoisomerases The topoisomerases The SSBPThe SSBP The polymerases I y IIIThe polymerases I y III The primaseThe primase The ligaseThe ligase

How does it happen?How does it happen?1. STRANDS ARE SEPARATED.1. STRANDS ARE SEPARATED.

The The helicasehelicase unwinds the double unwinds the double helix, separating both strands by helix, separating both strands by breaking the H bonds between breaking the H bonds between complementary bases. Next, the complementary bases. Next, the SSBPsSSBPs hold each strand in place. hold each strand in place. Why?Why?

The The DNA DNA polymerase IIIpolymerase III is the enzyme that, is the enzyme that, according to the sequence in the template brings according to the sequence in the template brings the complementary nucleotides adding them to the complementary nucleotides adding them to form a new strand.form a new strand.

Example:Example:Sequence in the template strand:Sequence in the template strand: A-T-T-C-C-G-A-T- … A-T-T-C-C-G-A-T- …

Complementary new strand formed: Complementary new strand formed: T-A-A-G-G-C-T-A- …T-A-A-G-G-C-T-A- …

REMEMBER COMPLEMENTARY BASE PAIRING: REMEMBER COMPLEMENTARY BASE PAIRING: A-T & C-G.A-T & C-G.

This happens in both template strands so that two “models” This happens in both template strands so that two “models” are being copied at the same time. However, ….are being copied at the same time. However, ….

2. SYNTHESIS OF NEW STRANDS

HOWEVER, there is a tiny detail HOWEVER, there is a tiny detail to take into account…to take into account…

REMEMBER THE DNA REMEMBER THE DNA STRUCTURE.STRUCTURE.

Strands are named according Strands are named according to the last free group bound to the last free group bound to the pentose in each end: to the pentose in each end: either a phosphate in C5’ or either a phosphate in C5’ or a OH in C3’. a OH in C3’.

Therefore strands are named Therefore strands are named as 5’ 3’ as 5’ 3’

and its complementary: and its complementary: 3’ 5’.3’ 5’.

DNA strands are said to be DNA strands are said to be ANTIPARALLEL.ANTIPARALLEL.

How to copy in opposite directions: How to copy in opposite directions: THE LEADING STRAND AND THE LEADING STRAND AND

THE LAGGING STRANDTHE LAGGING STRAND

SolutionSolution: first one strand is copied in one sense : first one strand is copied in one sense and next the other is copied in the opposite one.and next the other is copied in the opposite one.

The strand that gets copied first is known as the The strand that gets copied first is known as the leading strandleading strand and the second that gets copied and the second that gets copied next is known as the next is known as the lagging strandlagging strand. .

So what is the difference between the So what is the difference between the leading leading strandstrand and the and the lagging strandlagging strand? ? SimpleSimple: which one : which one is copied first. is copied first.

ProblemProblem: Polymerase III can only add nucleotides in the olymerase III can only add nucleotides in the direction 5’ – 3’direction 5’ – 3’ . This means that the new strand will be formed in the direction 5’ – 3’. So, where to start?

3. 3. HOW IS EACH STRAND COPIEDHOW IS EACH STRAND COPIED??Since the DNA polymerase III Since the DNA polymerase III

acts in the sense 5’—3’, acts in the sense 5’—3’, the strand that has a free the strand that has a free OH (in the sense OH (in the sense 3’—5’) 3’—5’)

will be acting as the will be acting as the leading strand leading strand .*.*

The leading strand is copied The leading strand is copied continuously.continuously.

The other strand (5’- 3’ The other strand (5’- 3’ sense) is copied next. sense) is copied next. Since it is in the opposite Since it is in the opposite direction, the polymerase direction, the polymerase will go slower and in will go slower and in chunks.chunks.

The lagging strand is copied The lagging strand is copied discontinuously in discontinuously in segments called segments called Okazaki Okazaki fragmentsfragments

*Pay attention to the templates (in blue): where replication starts and in what direction.

4. What is the DNA polymerase III 4. What is the DNA polymerase III for?for?

DNA polymerase III DNA polymerase III always acts in the 5’—always acts in the 5’—3’ sense3’ sense but it needs a but it needs a small segment of double small segment of double strand to start adding strand to start adding nucleotides.nucleotides.

This “starter fragment” This “starter fragment” known as known as primerprimer consists of RNA and is consists of RNA and is made by an enzyme made by an enzyme called called primaseprimase. .

Once the primer is in Once the primer is in position, polymerase III position, polymerase III just makes a new strand just makes a new strand adding complementary adding complementary nucleotides.nucleotides.

This takes place both in the This takes place both in the leading and lagging leading and lagging strands.strands.

So far …So far … In the leading strand, in the 3’—5’ sense, the DNA In the leading strand, in the 3’—5’ sense, the DNA

polymerase III adds complementary nucleotides polymerase III adds complementary nucleotides continuously to make a new strand starting with a RNA continuously to make a new strand starting with a RNA primer.primer.

On the other hand, in the lagging strand (in the 5’—3’ On the other hand, in the lagging strand (in the 5’—3’ sense) replication is discontinuous. The DNA polymerase III sense) replication is discontinuous. The DNA polymerase III adds complementary nucleotides as well, starting with a adds complementary nucleotides as well, starting with a RNA primer, but in chunks called the Okazaki fragments.RNA primer, but in chunks called the Okazaki fragments.

5. On we go …5. On we go …

Once the Okazaki fragments are synthesised on the Once the Okazaki fragments are synthesised on the one hand and the new continuous strand on the one hand and the new continuous strand on the other, there’s the final proof-reading to check if other, there’s the final proof-reading to check if any mistakes were made and the removal of the any mistakes were made and the removal of the RNA primers, which are no longer necessary.RNA primers, which are no longer necessary.

This is done by the enzyme This is done by the enzyme DNA DNA polymerase Ipolymerase I, , that: that:

Removes the RNA primersRemoves the RNA primers..

Adds a few more nucleotides to the Okazaki Adds a few more nucleotides to the Okazaki

fragments, elongating themfragments, elongating them..

Checks that newly synthesised strands are error-Checks that newly synthesised strands are error-

freefree (correct base pairing and no mismatches). (correct base pairing and no mismatches).

6. Finally…6. Finally…The only thing left is to join all the Okazaki fragments and the free The only thing left is to join all the Okazaki fragments and the free

nucleotides in the continuous strand. nucleotides in the continuous strand.

This is done by the This is done by the ligaseligase, which forms phosphodiester bonds between , which forms phosphodiester bonds between

the P- group of the first fragment and the OH from the following the P- group of the first fragment and the OH from the following

fragment.fragment.

Understood?Understood?Check the ends in both Check the ends in both

template strandstemplate strands

WARNING! To simplify diagrams, in books 2 DNA pol III are usually drawn making the new strands, but in fact it is only one.

Some animationsSome animations

http://www.youtube.com/watch?v=xnT_OuAYfvY

DNA song

http://www.youtube.com/watch?v=teV62zrm2P0&feature=related

DNA replication 1

http://www.youtube.com/watch?v=hfZ8o9D1tus

DNA replication 2