lecture 25_dna replication

8/13/2019 Lecture 25_DNA Replication

1/111

MBBS 2011 Batch

DNA Replication


2/111

Central Dogma of Biology


3/111

DNA Replication

The pivotal role of DNA in information

transfer in living cells, is called the central

dogma which includes three major steps in

the processing of genetic information.

The first is replication, the copying of

parent DNA to form daughter DNAmolecules having nucleotide sequences

identical to those of the parent DNA


4/111

The second step is transcription, the process in whichparts of genetic message in DNA are rewritten in the

form of RNA

The third step is translation in which the genetic messagecoded by RNA is translated by the ribosomes into the

protein structure

This flow of genetic information from DNA to RNA to

protein is called the central dogma of molecular biology


5/111

DNA is a major store of genetic information.

To transfer this genetic information from a

parent cell to a daughtercell during cellular

reproduction, the DNA must be duplicated.

The duplication or synthesis of DNA is

called replication


6/111


7/111

Proposed Models of DNA Replication In the late 1950s, three different mechanisms were proposed for the

replication of DNA Conservative model

Both parental strands stay together after DNA

replication Semiconservative model

The double-stranded DNA contains one parental

and one daughter strand following replication Dispersive model

Parental and daughter DNA are interspersed in

both strands following replication


8/111

How does DNA replicate? Possibilities:


9/111

DNA Replication

The mechanism by which DNA is replicated is consideredsemi-conservative

Semi-conservative replication: Half of the originalparent DNA molecule is conserved in each of the daughtermolecules.

This allows for the parent DNA to serve as a template for

generating the daughter DNA molecules Half of the replicated DNA strand is old and the

other half is new


10/111

Predictions of the Meselson-Stahl Experiment

Semiconservative replication

DN Replication

15N 15N 15N 15N

+

14N 14N 15N 15N

+

14N 14N14N14N14N14N

++1 gen. 1 gen.

All heavy Half intermediate,

half light

All intermediate


11/111


12/111

What Meselson and Stahl observed

Density of DNA decreases until one generation

time when it is halfway between the density of

totally heavy & totally light DNA; it is a hybridhalf new & half old

After 2 generation times, half of the DNA is

totally light & half is hybrid (half light, half heavy)


13/111

While semiconservative replication continues,original heavy parental strands remain intact &

present in hybrid DNA molecules, but they

occupy a smaller & smaller percentage of totalDNA

With time, the vast majority of DNA present is

fully light with 2 light strands


14/111


15/111

newnew old old

DNA Replication

Semi-Conservative


16/111

Base pairing During Replication Each old strand serves as a template for the new

complementary strand


17/111

Prokaryotic DNA replication Replication in prokaryotes is much better

understood than replication in eukaryotes.

Thebasic requirements and components of

replication are the same for prokaryotes as foreukaryotes.

Therefore, an understanding of how prokaryotes

replicate provides the understanding of howeukaryotes replicate


18/111

Basic requirements for replicationSubstrates

The four dNTPs

dATP

dGTP

dCTP

dTTP


19/111

Template

For DNA replication to occur, the two chains

have to unwind and separate.

The separated strands serve as template for thesynthesis of the new daughter strands

A template is required to direct the addition of

the appropriate complementary nucleotide to thenewly synthesized DNA strand


20/111

Enzymes

The DNA synthesis is catalysed by enzymes calledDNA dependent DNA polymerases. They are

called DNA dependent as they require DNAtemplate. They are more commonly called DNApolymerases.

These polymerases, which are required for:

DNA chain elongation DNA repair (5 to 3 exonuclease activity) and

Proof reading (3 to 5 exonuclease activity)


21/111

types of DNA polymerases DNA polymerase I: This enzyme completes chain

synthesis between Okazaki fragments on thelagging strand

DNA polymerase II: is mostly concerned withproof reading and DNA repair

DNA polymerase III: this enzyme functions at

replicative fork , catalyzing leading and laggingstrand synthesis


22/111

5 DNA polymerases in Eukaryotes

At least 5 DNA polymerases exist in eukaryotic cells,, ,,,Prokaryotic Eukaryotic

I Gap filling&Okazaki

fragment synthesisII DNA proofreading&repair

DNA repair mt DNA synthesis

III Leading &laggingstrand synthesis


23/111

Enzymes Required for Replication

Helicase: Melts or opens up the doublestrand so that the DNA is single stranded

Primase: Adds an RNA primer so that

DNA synthesis can begin

Ligase:Joins together small newly

synthesized pieces of DNA called Okazakifragments


24/111

Primer

The primer is a short piece of RNA, some10 nucleotides in length formed by DNA

dependent RNA polymerase, known as

primase, which synthesizes primer ( in a

5 to 3 direction) using DNA as a template


25/111

DNA polymerase initially adds adeoxyribonucleotide to the 3-hydroxyl

group of the primer and then continues to

add deoxyribonucleotides to the 3 end of

the growing strand


26/111

DNA is synthesized

5 to 3

Energy for

synthesis comes

from the removal of

the two phosphates

of the in comingnucleotide

DNA Replication


27/111

Stages of Replication

Devided into 3 stages

Initiation

ElongationTermination


28/111

Initiation

involves unwinding (separation) of twocomplementary DNA strands and formationof replicating fork

In prokaryotic orgnanisms, DNA replicationstarts at a particular DNA sequence, a sitecalled the origin of replication, ori

In eukaryotes replication begins at multiplesites composed almost exclusively of A-T

base pairs


29/111

Enzymes/proteins required for

initiation of replication of DNA

Dna A protein: opens duplex at a specificsite in ori

Dna B protein (helicase): unwinds DNA

Single stranded binding protein

(SSB): Binds single stranded DNA and

stabilizes the separated strand and preventsreannealing(renaturation)of DNA


30/111

DNA topoisomerases (I and II) or DNA

gyrase: relieves stress generated by

unwinding of DNA by helicaseDNA primase: initiates synthesis of RNA

primer


31/111

Steps in Initiation

First Dna A protein recognises andbinds to the ori of the DNA and

successively denatures the DNADna B protein (helicase) then binds to

this region and unwinds the parental

DNA, and form a V where activesynthesis occurs. This region is called

the replicating fork


32/111


33/111


34/111


35/111

DNA Replication

Since DNA is antiparallel, synthesis occursin opposite directions

One strand in continuously synthesized -

leading strand (53)

The other is synthesized in short

discontinuous strands - lagging strand(35)

Because of this DNA synthesis is called

Semidiscontinuous


36/111

DNA

Replication


37/111


38/111

The stress produced by supercoiling (due tounwinding by helicase) is released by

topoisomerasesby cutting either one or

both DNA strands

The SSB stabilizes the separated strands and

prevents their reassociation


39/111

Once the strands are sufficiently separated,binding of primase takes place. The primase

forms a complex with proteins known as

primosome which can recognize a specificsite of DNA where RNA primer synthesis

occur.

Primosome is formed at each of the

replicating forks.


40/111


41/111

Elongation

Once each RNA primer has been laid down,two DNA polymerase III complexes are

assembled, one at each of the primed sites.

Because of the antiparallel nature of the two

strands, the synthesis of DNA along the two

strands is different


42/111

The DNA chain which runs in the 35direction is copied by polymerase III in the

53 direction as a continuous strand,

requiring one primer.

This new strand is known as the leading

strand


43/111

The DNA chain which runs in the 53direction is copied by polymerase III in the

53 direction as a discontinuous manner

because synthesis can only proceed in the 5to 3 direction.

This new strand is known as the lagging

strand.


44/111

This also implies the need for numerousRNA primers at specific intervals followed

by synthesis of segments of DNA.

These segments are referred to as Okazaki

fragments (after their discoverer), are 1000

to 2000 nucleotides long.


45/111


46/111


47/111

Upon completion of lagging strand synthesisthe RNA primers are removed fromfragments by DNA polymerase I and

replaced with DNA in the gaps that areproduced by removal of the primers.

The resulting fragments are then joined by

the action of DNA ligase. DNA ligase sealsthe single stranded nick between Okazakifragments on the lagging strands


48/111


49/111


50/111

DNA ligase seals the gaps between Okazaki fragments with aphosphodiester bond (Fig. 3.7)


51/111

Termination

Termination sequences e.g., ter directtermination of replication.

A specific protein ter binding protein, binds

these sequences, and prevents the helicase

(Dna B protein) from further unwinding of

DNA and facilitates the termination of

replication


52/111


53/111

All three polymerases have 3 to 5exonuclease activity ( proofreadingactivity).

Pol I and II are known to excise erroneousnucleotides before the introduction of thenext nucleotide.

This process is known as proofreading.Theerror ratio during replication is thus kept ata very low level.


54/111


55/111

Eukaryotic DNA replication

DNA replication in eukaryotic organisms

resembles that in prokaryotic cells and

proceeds by a mechanism similar to that ofprokaryotic replication but is not identical.


56/111

Replication in Eukaryotes: Initiation

Higher organisms' cells have much more DNA

than bacteria & replicate DNA at much slowerrates; thus, they initiate replication at many

sites rather than just one as with the circular

chromosome ofE. coli

DN Replication


57/111


58/111


59/111


60/111

Mitochondrial DNA shows a much higherrate of mutation than nuclear DNA because

polymerase , which copies mt DNA has

no exonuclease activity


61/111

Reverse transcription

Reverse transcriptase uses RNA as a templateto synthesis DNA. It is therefore anRNA dependent DNA polymerase

Retroviruses carry RNA as their geneticmaterial and can synthesise double strandedDNA from their genomic RNAby a process

known as reverse transcription.An example of retrovirus is the human

immunodeficiency virus (HIV) which causes

AIDS.


62/111

DNA Replication


63/111

DNA Replication: Fast & Accurate!

It takes E. coli


64/111

Summary

1. The origin of replication is identified. Then theparental DNA unwinds to form replication fork

2. RNA primer complimentary to the DNA template

is synthesized by RNA primase

3. DNA synthesis is continuous in the leading

strand ( towards replication fork) by DNA

polymease III

4. DNA syntehsis is discontinuous in the laggingstrand (away from the fork), as Okazaki

fragments


65/111

5. Elongation: In both strands, synthesis isfrom 5 to 3 direction

6. Then the RNA pieces are removed; the gapsgenerated are filled by deoxyribonucleotidesby DNA polymerase I and th epieces areligated (joined) by DNA ligase

7. Proof reading is done by the DNA

polymease


66/111

Transcription (RNA synthesis)

Transcription is defined as the synthesis ofRNA molecule using DNA as a template,

that results in the transfer of the

information stored in double stranded DNAinto a single stranded RNA, which is used

by the cell to direct the synthesis of proteins


67/111


68/111

Cellular RNAs

Include1. Messenger RNA (mRNA)

2. Ribosomal RNA (rRNA)

3. Transfer RNA (tRNA) and4. Several small nuclear RNAs (snRNAs)

All are transcribed from DNA. The first

three RNAs are involved in proteinsynthesis and sn-RNA is involved inmRNA splicing


69/111

Replication&Transcription

The process of DNA and RNA synthesis aresimilar in that:

1. They involve the general steps initiation,

elongation and termination

2. Synthesis occurs in 5 3 direction

3. FollowsWatson-Crick base pairing rules


70/111


71/111

Requirements

TemplateA single strand of DNA acts as a template to

direct the formation of complementary

RNA during transcription.The strand that is transcribed into RNA

molecule is referred to as the template

strand of the DNA.The other DNA strand is referred to as the

coding strand of the gene.


72/111

Substrates

The substrates for RNA synthesis are thefour ribonucleoside triphosphates,

ATP

GTP

CTP

UTP


73/111

RNA polymerase

DNA dependent RNA polymerase called RNApolymerase is responsible for the synthesis of

RNA in 5 to 3 direction, using DNA template

Prokaryotic RNA polymerase

Prokaryotes have single RNA polymerase that

transcribes all three RNAs i.e.,

m-RNA

r-RNA and

t-RNA

RNA polymerase contains four subunits


74/111

RNA polymerase contains four subunits(2 , , ) which form the core enzyme. The

active enzyme, the holoenzyme contains coreenzyme and a fifth subunit called sigma ()factor

The sigma factor is required for binding of thepolymerase to specific promoter regions ofDNA template.

RNA polymerase is a metalloenzyme, containgzinc molecule


75/111

St g f t i ti


76/111

Stages of transcription

Transcription process is best understood inprokaryotes.

The description of RNA synthesis in

prokaryotes is applicable to eukaryotes eventhough the enzyme involved and regulatorysignals are different

RNA synthesis involves initiation

elongation and

termination


77/111

Initiation

Initiation involvesbinding of RNApolymerase to the DNA template at the

promoter site

Promoters are specific regions contained inthe DNA that are recognized by RNA

polymerase.

The size of the promoter region is variable.


78/111

In prokaryotes, the promoter region rangesfrom 20-200 bases

The core enzyme(2,,) alone can not

recognize the promoter regions.

The subunit is essential for this function

During binding of RNA polymerase to the


79/111

g g p y

template, the following sequence of events

occurs.

The subunit of RNA polymerase recognizesthe promoter sequence

RNA polymerase attaches to the promoter

region

RNA polymerase melts the helical structure

and opens the DNA

RNA polymerase initiates RNA synthesis on thedenatured single stranded DNA template


80/111

Unlike initiation of replication,transcriptional initiation does not require

primer

Promoter sequences are responsible fordirecting RNA polymerase to initiate

transcription at a particular point known as

start point or initiation site


81/111

In prokaryotes a single factor namely sigmafactor is needed to initiate transcription

The sigma factor enables the RNA

polymerase holoenzyme to recognize andbind tightly to the promoter sequences

In eukaryotes multiple factors are required

because of the diversity of promoters andcomplexity of their RNA polymerases


82/111

Thebinding of RNA polymerase to theDNA template results in the unwinding of

the DNA double helix

The enzyme then catalyses the formation ofphosphodiester bond between the first two

bases.

The first base is usually a purine nucleoside

triphosphate


83/111


84/111


85/111


86/111


87/111

-35 region

1. RNA polymerase binds first to theupstream side of the -35 sequence via a

site recognized by the subunit

2. Because of its huge size , the RNApolymerase then comes into contact

with the Pribnow box

3. Once bound to the Pribnow box, RNA

polymerase dissociates from the initial

recognition site


88/111

-35 -10

+1

TTGACA--------------TATAAT-------------Start

site


89/111


90/111


91/111

The second incoming NTP binds to the


92/111

The second incoming NTPbinds to theelongation site on the polymerase. The NTP

is chosen on the basis of its ability toHydrogen-bond with the complementary

base on the DNA

RNA polymerase covalently bonds the firstand second bases

The first base dissociates from the initiation

site, and initiation is then completeThe presence of triphosphate moiety

suggests that RNA synthesis starts at 5 end


93/111

Most newly synthesised RNA chains carry ahighly distinctive tag on the 5 end : the first

base at that end is either pppG or pppA

The presence of the triphosphate moietysuggests that RNA synthesis starts at the 5end

RNA chains, like DNA chains grow in the53 direction


94/111


95/111


96/111


97/111


98/111


99/111


100/111


101/111

Rho independent termination

Involves a secondary structure, hair pin loopformed in the newly synthesised RNAwhich dislodges the RNA polymerase fromDNA template, resulting in the release ofthe transcript

In eukaryotes termination is less welldefined. It is believed that it is similar tothat of rho independent termination


102/111


103/111

Termination Signal.A termination signal found at the 3 end of

an mRNA transcript consists of a series of

bases that form a stable stem-loop structureand a series of U residues.

P i i l i


104/111

Post-transcriptional processing

Primary transcript made by RNApolymerase undergo further modification,

called post-transcriptional processing.

1. Cleavage of larger precursor RNA for theremoval of excess sequences from the

primary transcript by the action of

endonucleases for a smaller molecules


105/111


106/111

2. Splicing:Involves the removal of sequences

called introns (sequences that do not

code for proteins) from the primary

transcript and joining of other

sequences called exons (codingsequences) to each other to form

functional RNA


107/111


108/111


109/111


110/111

RNA synthesis: Transcription inhibitorsActinomycin D (dactinomycin)

Rifampin

-amanitin


111/111

lecture 25_dna replication

Documents