Chap 2 DNA Replication

8/19/2019 Chap 2 DNA Replication

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NAR 2007 DNA Replication 1

Chapter 2

DNA Replication



NAR 2007 DNA Replication

2

The Gene is the Fundamental Unit ofHeredity

The unit of heredity is known as a gene. Each gene is

responsible for a single inherited property or characteristic of

the organism.

Inheritance of Genetic Information

Since each cell needs a complete set of genes it is necessary

for the original cell to duplicate its genes before di!iding.

"ecause the genes are made of DNA and make up the

chromosomes this means that each chromosome must

be accurately copied. #pon cell di!ision both daughter

cells will recei!e identical sets of chromosomes each with a

complete set of genes.




3

eplication of DNA

Replication of the DNA in molecular terms

means that the DNA of the original or motheris duplicated to gi!e t!o identical copies.

This process is known as replication.

#pon cell di!ision each of the descendants

gets one complete copy of the DNA. The

original genes of the mother cell are on a

double stranded DNA molecule so the $rststep in replication is to separate the

t!o strands of the DNA double heli%.




4

E%periments in the &'()s showedthat DNA is the hereditarymaterial

Scientists raced to determine thestructure of DNA

&'(* + ,atson and -rick proposedthat DNA is a double heli%




5

The ne%t step is tobuild a comple+mentary strand oneach of the two

original strands.Since A only pairs!ith T and since Gonly pairs !ith Cthe seuence of

each stranddictatesthe seuence of itscomplementarystrand.

* bonds/

bonds




6

,e now ha!e two double stranded DNA

molecules both with seuences identical to the

original one. 0ne of these daughter moleculeshas the original left strand and the other

daughter has the original right strand.

This is known as semiconser"ati"ereplication of the progeny conser!es half of

the original DNA molecule.

A # T $ C # G%




7

&emiconser"ati"e eplication

1arentalstrands

1arentalstrandsseparate

1arental strandrecei!es new strand ofDNA 2& set + one oldand one new strand3

'2

(

)

Strand & is identical to strand *

Starnd / is identical to strand 4

Strand & is complementary to strand/

Starnd * is complementary to strand

4




8

Each parent strand

remains intact

E!ery DNA molecule ishalf 5old6 and half 5new6




9

7ow Does a Double 7eli% Separate into Strands8

"ecause the two strands forming a DNA molecule

are held together by hydrogen *onding and

twisted around each other to form a dou*le

heli+ they cannot simply be pulled apart.

,orse still the DNA inside a cell is

also supercoiled to pack it into a small space.

"efore separating the strands both the supercoils

and the dou*le heli+ must *e un!ound.




10

This is done in two stages 9

&. :irst the supercoils are unwound by an en;ymeknown as DNA gyrase ,DNA topoisomerase-.

The gyrase cuts both strands of double stranded

DNA to gi!e a double stranded break.

7owe!er it keeps hold of all of the cut ends. Thetwo hal!es of the gyrase then rotate relati!e toeach other and the ends are re<oined. Thisuntwists the supercoils. Each rotation costs thecell a small amount of energy.

7ow Does a Double 7eli% Separate into Strands8




11

Double stranded breaks done by DNA gyrase will relie!esupercoiling but will not pull the strands apart because the strandsare still held by the hydrogen bonds between the bases. 0nceuntwisted the ends are re<oined.

Action of DNA Gyrase




12

/. 0nce the supercoils ha!e been untwisted the

double heli% is unwound by the en;yme DNA

helicase. 7elicase does not break the DNA

chain it simply disrupts the hydrogen

bonds holding the base pairs together.

H th . t l &t d f DNA / t A t0




13

Ho! are the .arental &trands of DNA /ept Apart0

The two separated strands of the parental DNA

molecule are complementary to each other.-onseuently all of their respecti!e bases arecapable of pairing o= and binding to each other.

>n order to manufacture the new strands the two

original strands despite their desire to clingtogether must somehow be kept apart.

This is done by means of a special

?di!orce? protein which binds to the unpairedsingle stranded DNA and pre!ents the two parentalstrands from getting back together. This is knownas &ingle &trand 1inding protein ,&&1-




14

3

&ingle strand *inding protein

3

(3

(3

&ingle4strand *indingprotein

helicase

Directionofeplication




15

5a6ing a Ne! &trand of DNA

The critical issue in replication is the base pairing of A

with T and of G with C. Each of the separated parental

strands of DNA ser!es as a template strand for the

synthesis of a new complementary strand.

The incoming nucleotides for the new strand recogni;e

their partners by *ase pairing and so are lined up on

the template strand. Actually things are a bit more

complicated.




16

1ase .airing duringeplication

Each old strandser!es as thetemplate forcomplementarynew strand




17

Although hydrogen bonding alone would matchbases correctly 27783 of the time this is not

good enough.

The en;yme that links the nucleotides known as

DNA polymerase III or pol III can also sense if

the base are correctly paired. >f not the

mismatched base pair is re<ected.




18

"ase+pairing of

DNA

@

T

A

A

-

-

-

T

T

@

A

T

-

@

@

@

T

A

A

-

ne!

ne!




19

9H(3

G9:ING&TAND

T

G

A

A

G

A

C

A

T A

C

T

G

T

3

(39H

5’

T;5.<AT;&TAND

phosphate

9H

DNA polymerase >>>

DNA .9<=5;A&; III 5A/ING DNADNA Polymerase III Pol (III) enzyme thatmakes most of the DNA when chromosome are

replicated




20

Energy for

strand

assembly is

pro!ided by

remo!al of two

phosphate

groups from

free nucleotides

A Closer Look at

Strand Assembly




21

DNA polymerase III

The nucleotides are then <oined together by theen;yme. This DNA polymerase has two subunits.

i. 0ne of these is the synthetic su*unit and

is responsible for manufacturing new DNA.

ii. The other subunit is shaped like a doughnut

and slides up and down like a curtain ring on

the template strand of DNA. This >sliding

clamp> su*unit binds the synthetic subunit tothe DNA.




22

Dna .olymerase III ? The &liding Clamp

SSBs

9riginal strand ofDNA

Pol III- sliding clamp

subunit

.ol III4 syntheticsu*unit

Newly synthesized DNA




23

&liding clamp su*unit




24

&ynthesis Al!ays Goes from @ to (3

As you know nucleotides ha!e three components9a. a phosphate group

*. sugar and

c% the *ase%

>n DNA the sugar is deo+yri*ose and is <oinedto the base at position '@ and to thephosphate group at position @.

2The carbon atoms of the deo%yribose sugar arenumbered with prime marks to distinguish them

from those of the base which ha!e plain numbers3




25

1A&;

1’

H

H

O

.hosphate494CH2

!’

"’

#’

$’

Ne+t nucleotide is

oined here




26

'2(

)

(

1A&;

1’

H

H

O

!’

"’

#’

$’H2C

9

.

9

9H

49

H2O

07




27

Direction of DNA &ynthesis

,hen a new nucleotide is added it is <oined !ia its own

phosphate group on position @ to the (@ position as

indicated by the arrow.

New DNA strands always start at the ( end and grow in

the * direction. >n fact all nucleic acids whether DNA or

RNA are always made in the ( to * direction. -

7owe!er DNA is normally double stranded and it happens

that the two strands run in opposite directions that is if

one goes (B to *B then its complementary partner will run

from *B to (B. The strands are said to be anti4parallel.

Dou*le stranded DNA is antiparallel




28

3

(3 3

(3

American Cames ,atson <oined with:rancis 7. -. -rick in England to

work on structure of DNA. ,atsonand -rick recei!ed the Nobel 1ri;e in&'/ for their model of DNA.

#sing information generated by-harga= and :ranklin ,atson and

-rick built a model of DNA as doubleheli% sugar+phosphate molecules onoutside paired bases on inside.

Complementary *ase pairing isthe paired relationship *et!een

purines and pyrimidines in DNAsuch that A is hydrogen+bonded to Tand @ is hydrogen+bonded to -.






29

AA -- T T T T AA -- @@ --

@@ T T @@ AA AA T T @@ -- @@

$’ "’

"’ $’


--




30

The Replication :ork >s ,here the Action >sF

The replication fork is the total structure in the region

where the DNA molecule is being duplicated.

>t includes the swi!el where the DNA is being twisted byDNA gyrase the helicase following right behind andthe stretches of single stranded DNA held apart by the

single strand *inding protein.

>t also has two molecules of DNA polymerase III whichare busy making two new strands of DNA. Since DNA is

always made in the (B to *B direction and since thetwo strands of double helical DNA are antiparallel thismeans that during DNA replication the two new strandsmust be synthesi;ed in opposite directions.




31

5’

5’

5’

5’

3’

3’

3’

3’

5’

3’

SSB DNA helicase

%aggingst&and

%eadingst&and

The eplicationFor6




32

The eplicationFor6

a Nucleoside triphosphates ser!e asa substrate for DNA polymerase

according to the mechanism shownon the top strand. Each nucleosidetriphosphate is made up of threephosphates 2represented here byyellow spheres3 a deo%yribose sugar2beige rectangle3 and one of four

bases 2di=erently colouredcylinders3. The three phosphates are <oined to each other by high+energybonds and the clea!age of thesebonds during the polymeri;ationreaction releases the free energy

needed to dri!e the incorporation ofeach nucleotide into the growingDNA chain. The reaction shown onthe bottom strand which wouldcause DNA chain growth in the * to( chemical direction does not occur

in nature.




33

The DNA replication for6 ? thee+planation

* DNA polymerases catalyse chain

growth only in the ( to * chemicaldirection but both new daughterstrands grow at the fork so adilemma of the &')s was how thebottom strand in this diagram wassynthesi;ed. The asymmetric nature

of the replication fork was recogni;edby the early &'G)s9 the Hleadingstrand grows continuously whereasthe Hlagging strand is synthesi;ed bya DNA polymerase through thebackstitching mechanism illustrated.

Thus both strands are produced byDNA synthesis in the ( to *direction.

Completing the <agging &trand




34

Although the leading strand <ust keeps getting longer

and longer the lagging strand is handicapped.

After the replication fork has passed by the >agging

strand is left as a series of short pieces with gaps in

between. These newly made pieces of DNA areknown as 96aBa6i fragments after their disco!erer

and must be <oined together to gi!e a complete

strand of DNA.

Completing the <agging &trand

Replication Fork Revisite




35

p




36

Gap inlaggingstrand

.ol I FillsGap !ithnucleotid

es

<igasesealsnic6

Ne!ly addednucleotides

9/AA/IFAG5;NT

9rigin

al DNAstrand

$’

$’

5’

"’

"’

"’

DNA.ol '

"’

"’

"’

$’

$’

Directionofsynthesis

Directionofsynthesis

DNA ligase

$’

"’

"’

$’

9ne nic63remains

'AP

Nic63 emains

Ne! 34(3 *ond




37

This is accomplished by two en;ymes working in

succession9 DNA polymerase I and DNA ligase.DNA polymerase > $lls in the gaps and DNA ligase <oins the gaps.

DNA polymerase > was disco!ered before DNA

polymerase >>> hence the numbering.

"oth DNA polymerase > and DNA ligase ha!eimportant uses in genetic engineering.

&tarting a Ne! &trand




38

&tarting a Ne! &trand

#p to now we ha!e assumed that we ha!e strandsof DNA with free ends that can be elongated byDNA polymerase. "ut how do we get a new strandstarted8

Although the leading strand only needs to bestarted once the lagging strand is made in shortsections and we need to start again e!ery timewe make a new 96aBa6i fragment.

-uriously DNA polymerase cannot start a ne!strand *y itself$ it can only elongateF




39

New strands are started with short stretch not

of DNA itself self but of RNAF

These short NA pieces known as primers

and the en;yme that starts synthesis of new

chains by making the RNA primers is calledprimase.

So e!ery time a new fragment of DNA is made

primase sneaks in and lays down a short RNAprimer to get things going. 0nly then can DNA

polymerase get to work elongating the strand.

ili th DNA i t H li




40

ecoiling the DNA into a Heli+

As the two new strands of DNA are synthesi;edtwo double DNA molecules are produced eachwith one old and one new strand.

0nce the replication fork has mo!ed past the

double stranded DNA molecule automaticallyrewinds into a heli%.




41

;nBymes in"ol"ed in DNA eplication4 details

T!o DNA polymerase molecules are acti!e at the fork at

any one time. 0ne mo!es continuously to produce the newdaughter DNA molecule on the leading strand whereas theother produces a long series of short H0ka;aki DNAfragments on the lagging strand.

"oth polymerases are anchored to their template bypolymerase accessory proteins in the form of a slidingclamp and a clamp loader.

A DNA helicase powered by AT1 hydrolysis propels itself

rapidly along one of the template DNA strands 2here thelagging strand3 forcing open the DNA heli% ahead of thereplication fork.




42

,DNA gyrase-




43

;nBymes4 details

The helicase e%poses the bases of the DNA

heli% for the leading+strand polymerase to copy.DNA topoisomerase or DNA gyrase en;ymesfacilitate DNA heli% unwinding.

>n addition to the template DNA polymerasesneed a pre+e%isting DNA or RNA chain end 2aprimer3 onto which to add each nucleotide.

:or this reason the lagging strand polymerasereuires the action of a DNA primase en;ymebefore it can start each 0ka;aki fragment.




44

Iore stu= on the en;ymes for replication

The primase produces a !ery short RNAmolecule 2an RNA primer3 at the ( end of each

0ka;aki fragment onto which the DNA

polymerase adds nucleotides

:inally the single+stranded regions of DNA at

the fork are co!ered by multiple copies of a

single4strand DNA4*inding protein whichhold the DNA template strands open with their

bases e%posed.

Jeading strand




45

>n the folded fork structure shown in the inset the

lagging+strand DNA polymerase remains tied to

the leading+strand DNA polymerase. This allows

the lagging+strand polymerase to remain at thefork after it $nishes the synthesis of each 0ka;aki

fragment.

Jaggingstrand

Jeadingstrand

Jeading strandDNA pol >>>

Jagging strandDNA pol >>>

DNAgyraseDNA

helicase

DNAhelicase




46

1inary Ession in *acteria

Replication of chromosomal DNA in bacteriastarts at a speci$c chromosomal site called

the origin and proceeds *idirectionally until

the process is completed.

,hen bacteria di!ide by binary $ssion after

completing DNA replication the replicated

chromosomes are partitioned into each of the

daughter cells.




47

The origin regions speci$cally andtransiently associate !ith the cellmem*rane after DNA replication has beeninitiated leading to a model wherebymembrane attachment directs separation ofdaughter chromosomes 2the replicon model3.

These characteristics of DNA replicationduring bacterial growth ful$ll thereuirements of the genetic material to be

reproduced accurately and to be inherited byeach daughter cell at the time of celldi!ision.




48

;.<ICATI9NF9/,*idirectionalreplication-

9IGIN

DNA eplication in 1acteria



Membrane growth movesDNA molecules apart

DNA replication completedParent DNAmolecule

DNA copy

DNA replication

begins

Bacterium before DNAreplication

Bacterialchromosome

New membrane and cellwall deposited

Cytoplasm divided into two.

E.coli undergoing*inary Ession

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Chap 2 DNA Replication

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