Cap 11 biologia

8/20/2019 Cap 11 biologia

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1

Chapter 11

Lecture and

Animation Outline

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Chapter 11

Nucleic Acid Structure, DNA Replication,

and Chromosome Structure

Biochemical Identification of the enetic !aterial

Nucleic Acid Structure

An O"er"iew of DNA Replication

!olecular !echanism of DNA Replication

!olecular Structure of #u$ar%otic Chromosomes

2

ey Concepts&


3/70

hat is the &enetic material'

/our criteria necessary or genetic material&1( Information

)( Replication*( +ransmission

( Variation

0ate 1233s 4 -iochemical -asis of heredit% postulated

5esearchers "ecame con(inced that chromosomes carrythe genetic inormation

1673s to 1683s 4 scientists e%pected the protein portion o

chromosomes would turn out to "e the genetic material

3

Biochemical Identification

of the enetic !aterial


4/70

riffith.s -acterial transformation

0ate 1673s 4 /rederic$ riffith was wor+ing withStreptococcus pneumoniae "acteria

Two strains o S. pneumoniae&

!trains that secrete capsules loo+ smooth 0S

and inections are atal in mice

!trains that do not secrete capsules loo+ rou&h 0R and

inections are not atal in mice

The capsule shields the -acteria rom the immune

system, so they sur(i(e in the "lood

4



5/70

5


+reatment Result Conclusion

Control2

In3ected li"in&

t%pe R -acteria

into mouse(

) +%pe R cellsare -eni&n(

Control2

In3ected heat4

$illed t%pe S

-acteria

into mouse(

* 5eat4$illedt%pe S cells

are -eni&n(

1 +%pe S cellsare "irulent(

Control2

In3ected li"in&

t%pe S -acteria

into mouse(

Virulent t%pe S

strain in dead

mouse.s -lood

Li"in& t%pe

R cells ha"e-een

transformed

into "irulent

t%pe S cells

-% a

su-stance

from the

heat4$illed

t%pe S cells(

In3ected li"in&

t%pe R and

heat4$illed

t%pe S -acteria

into mouse(


6/70

!mooth strains )!* with capsule are atal9 roughstrains )5* without capsule are not

I mice are in:ected with heat4$illed t%pe S, they

sur(i(e )"ecause "acteria are dead*

Howe(er, mi6in& li"e R with heat4$illed S +ills

the mouse

;lood is ound to contain li"in& t%pe S -acteria

nown as transformation

6


7/70

How is this possi"le<

enetic material had -een transferred

rom the heat-+illed type ! "acteria to the li(ing

type 5 "acteria This ga(e them the capsule4secretin& trait and

was passed on to their ospring

hat was the -iochemical -asis o thistransorming principle< =t the time there was no

way to +now

7


8/70

A"er%, !acLeod, and !cCart% 7sed

8urification !ethods to Re"eal +hat

DNA is the enetic !aterial 1683s 4 interest in inding "iochemical "asis o "acterial

transormation

'nly purified DNA rom type ! could transorm type 5 ;ut, puriied >?= might still contain traces o

contamination that may "e the transorming principle

=dded DNase, RNase and proteases

5?ase and protease had no eect

hen >?ase was added, no transormation too+ place

!urprising conclusion& DNA is the &enetic material


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1 8urif% DNA from a t%pe S strain(+his in"ol"es -rea$in& open cells

and separatin& the DNA awa% from

other components -%

centrifu&ation(

DNase

RNase

8rotease

9 +%pe R cells

Control

A B C D #

A B C D #

!i6 the DNA e6tract with t%pe R

-acteria( Allow time for the DNA

to -e ta$en up -% the t%pe R cells,

con"ertin& a few of them to t%pe S(

Also, carr% out the same steps -ut

add the en:%mes DNase, RNase, or

protease to the DNA e6tract, which

di&est DNA, RNA, and proteins,

respecti"el%( As a control, don.t add

an% DNA e6tract to some t%pe R cells(

Add

anti-od%9 DNA 9 DNA

9 DNase

9 DNA

9 RNase

9 DNA

9 8rotease

)


#6perimental le"el Conceptual le"el

5;8O+5#SIS A purified macromolecule from t%pe S -acteria, which functions as the &enetic material, will -e a-le to con"ert t%pe

R -acteria into t%pe S(


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A

B

C

D

#

Control

= +5# DA+A

DNA e6tract

DNA e6tract 9 DNase

DNA e6tract 9 RNase

DNA e6tract 9 protease

CONCL7SION DNA is responsi-le for transformin& t%pe R cells into t%pe S cells(

SO7RC# A"er%, O(+(, !acLeod, C(!(, and !cCart%, !( 1>( Studies on the Chemical Nature of the Su-stance Inducin&

+ransformation of 8neumococcal +%pes( Journal of Experimental Medicine ?>21*?@1=(

?

Centrifu&e

Remo"e t%pe R cells -%centrifu&ation( 8late the remainin&

-acteria 0if an% that are in the

supernatant onto petri plates(

Incu-ate o"erni&ht(

+%pe S cells

in supernatant

+%pe R cells

in pellet


11/70

5ershe% and Chase

16@7 4 studied a T7 (irus that inects Escherichia coli ;acterial (irus is +nown as -acteriopha&e or pha&e

Phage coat made entirely o protein

DNA ound inside capsid

11


DNA

8rotein

0a Schematic drawin& of +) -acteriopha&e

DNA

8ha&e head

0capsid

Sheath

+ail fi-er

+) &enetic

material -ein&

in3ected into

E. coli

E. coli cell

Base plate

0- An electron micro&raph of +) -acteriopha&einfectin& E. coli

= nm

© Aye o !cience$Photo 5esearchers, Inc.


12/70

12


* 7sin& a ei&er counter,determine the amount of radioacti"it% in the supernatant(

ei&er 0radioisotope

counter

#6periment 1 #6periment )

1

Bacterial cell Bacterial cell

Sheared empt% pha&e

E. coli cells wereinfected with*=S4la-eled pha&e

and su-3ected to

-lender treatment(

8ha&e DNA

*=S4la-eled sheared

empt% pha&e

E. coli cells wereinfected with*)84la-eled pha&e

and su-3ected to

-lender treatment(

*)84la-eled

pha&e DNA

) +ransfer to tu-eand centrifu&e(

Supernatant

has *=S4la-eled

empt% pha&e(

+ransfer to tu-e

and centrifu&e(

Supernatant has

unla-eled empt%

pha&e(

8ellet has

E. coli cells

infected with*)84la-eled

pha&e DNA(

8ellet has

E. coli cells

infected with

unla-eled

pha&e DNA(

+ o t a l i s o t o

p e i n s u p e r n a t a n t 0 C 1

A&itation time in -lender 0min

1

)

1 ) * = ?

+5# DA+A

#6tracellular *=S

#6tracellular *)8

Blendin& remo"es

of *=S from cells(

!ost of the *)8 0=

remains with intact cells(


13/70

0e(els o >?= !tructure&

1( Nucleotides 4 the "uilding "loc+s o >?= and 5?=

)( Strand 4 a linear polymer strand o >?= or 5?=

*( Dou-le heli6 4 the two strands o >?=

( Chromosomes 4 >?= associated with an array o

dierent proteins into a comple% structure

=( enome 4 the complete complement o genetic

material in an organism

13

Nucleic Acid Structure


14/70

14


Sin&le strand

Nucleotides

Dou-le heli6

DNA associates with

proteins to form a

chromosome(


15/70

DNA

/ormed rom nucleotides )=, G, C, T*

?ucleotides composed o

three components

8hosphate &roup 8entose su&ar

>eo%yri"ose

>?= B >eo%yri"onucleic =cid

Nitro&enous -ase

Purines 4 =denine )=*, Guanine )G*

Pyrimidines 4 Cytosine )C*, Thymine )T*

15

Base

8hosphate

Deo6%ri-ose


16/70

16

DNA nucleotidesCopyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.

0a DNA nucleotide

8hosphate

Deo6%ri-ose

Base

+h%mine 0+Adenine 0A

C%tosine 0Cuanine 0

N5)

O

5

5

N

N

N5)

N

5 5

5 5

N

5

N

N

O

N5)

5

N

N

N

5

N

5

5O5

55

OO

O @

C5)

O @

8

O 5

5

O

N

O

N5

8urines

0dou-le rin&

8%rimidines

0sin&le rin&

C5*


17/70

RNA

/ormed rom nucleotides )=, G, C, #*

?ucleotides composed o

three components

8hosphate &roup 8entose su&ar

5i"ose

5?= B 5i"onucleic =cid

Nitro&enous -ase

Purines 4 =denine )=*, Guanine )G*

Pyrimidines 4 Cytosine )C*, #racil )#*

17

Base

8hosphate

Ri-ose

H

O5


18/70

18

RNA nucleotidesCopyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.

0- RNA nucleotide

Ri-ose

7racil 07Adenine 0A

uanine 0

5

C%tosine 0C

N5)

O

5

5

N

N

5

5 5

5

5

5

N5)

N

N

5

N

N

O

N5)

5

N

N

N

5

N

O

O

N

N

8hosphate

Base

5

O5O5

55

OO

O @

C5)

O @

8O

5


19/70

!ugar car"ons are 1 to @

;ase attached to 1 car"on on sugar

Phosphate attached to @ car"on on sugar

19

Nucleotide num-erin& s%stem


55

5O5

55

OO

O @

O @=E

E 1E

)E *E

*

)1

=

8O

O

5

5

N

O

N

+h%mine

C5*

C5)

8hosphate

Deo6%ri-ose


20/70

Strands

?ucleotides are

co(alently "onded

8hosphodiester -ond

4 phosphate group lin+stwo sugars

Bac$-one 4 ormed rom

phosphates and sugars

;ases pro:ect away rom"ac+"one

ritten @ to

e%& @ 4 T=CG 4 20


O

5

5

N

O

O @

N

N

5

N

N

55

5

*E

)E *E

N

+h%mine 0+

Adenine 0A

BasesBac$-one

C5*

Su&ar 0deo6%ri-ose

O5

5

8hosphate

N5)

5 5

OO 8

O @

OO

O

=E

E

*E

8

=E C5)

C5)

5

5

5

O

1E

)E

O

O

N

N

55

5

55

O

OO 8

O @

5N

N

N

5

N

55

O

OO

O

8

=E

E 1E

)E *E

=E

E 1E

C%tosine 0C

uanine 0

8hosphodiester

lin$a&e

Sin&le

nucleotide

C5)

N5)

N5)

C5)

5

5

5

5

5

O

1E

)E *E

O

55

O @

=E

E

O @


21/70

Sol"in& the structure of DNA

16@, Fames Gatson and /rancis Cric$, proposed the

structure o the >?= dou"le heli%

atson and Cric+ used 0inus Paulings method

o wor+ing out protein structures using simple-all4and4stic$ models

Rosalind /ran$lin.s

H4ra% diffraction results

were crucial e(idence,

suggesting a helical structure

with uniorm diameter

21


22/70

22


H4ra%s diffracted -% DNA

onto photo&raphic plate

8attern represents the

atomic arra% in wet fi-ers

Get DNA fi-ers

H4ra% -eam


23/70

#rwin Char&off analyDed "ase composition

o >?= rom many dierent species

5esults consistently showed

amount o adenine )=* B amount o thymine )T*

amount o cytosine )C* B amount o guanine )G*

23

Base4pairin&


24/70

24


25/70

Gatson and Cric$

Put together these pieces o inormation

/ound -all4and4stic$ model consistent with

data

Dou-le4stranded heli6

Base4pairin&2 = with T and G with C

Eames atson, /rancis Cric+, and Maurice

il+ins awarded ?o"el PriDe in 16F7

5osalind /ran+lin had died and the ?o"el PriDe

is not awarded posthumously

25


26/70

Dou-le stranded

Antiparallel strands

Ri&ht4handed heli6

Su&ar4phosphate

-ac$-one

Bases on the inside

!ta"iliDed "y 54-ondin&

!peciic -ase4pairin&

13 nts per helical turn 26

/eatures of DNA


Bases

5%dro&en -ond

) nm

0a Dou-le heli6

=E end

*E end

Su&ar4phosphate

-ac$-one

One nucleotide

(* nm

*E end

=E end

Complete turn

of the heli6

*( nm


27/70


55

5

5

OOO 8

N

O 5

5

N

O

C5*

C5)

O

5N

N

N

5

N

5 5

5

55

OOO 8C5)

O

O

5N

N

N

5

N

5 5

5

55

OOO 8C5)

5 N

N

5

N

N

5 5

5

55

OOO 8C5)

5O

C%tosine

C%tosine

uanine

uanine

+h%mine

Adenine

O

55

N

N

55

5

55

OO

O

8 C5)

O @O

O

55

N

N

55

5O5

55

OO

O

8 C5)O

O

=E phosphate

*E h%dro6%l

0- Base pairin&


28/70

Char&off.s rule = pairs with T

G pairs with C

eeps width consistent

Complementar% DNA strands @ 4 GCGG=TTT 4

4 CGCCT=== 4 @

Antiparallel strands'ne strand @ to

'ther stand to @

28


29/70

Groo(es are re(ealed in

the space-illing model

!a3or &roo"e

Proteins "ind to aect genee%pression

!inor &roo"e

?arrower

29


!a3or &roo"e

!inor &roo"e

!a3or &roo"e

!inor &roo"e


30/70

0ate 16@3s 4 three dierent models were

proposed or >?= replication

Semiconser"ati"e !odel

Conser"ati"e !odel

Dispersi"e !odel

?ewly-made strands are daughter strands

'riginal strands are parental strands

30

An O"er"iew of DNA Replication


31/70

31

Semiconser"ati"e !echanismCopyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.

Second roundof replication

/irst roundof replication

Ori&inaldou-le heli6

8arental strand

Dau&hter strand

0a Semiconser"ati"e mechanism( DNA replication produces

DNA molecules with 1 parental strand and 1 newl% made

dau&hter strand(


32/70

32

Conser"ati"e !echanismCopyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.

Second roundof replication

/irst roundof replication

Ori&inaldou-le heli6

0- Conser"ati"e mechanism( DNA replication produces 1 dou-le

heli6 with -oth parental strands and the other with ) new

dau&hter strands(


33/70

33

Dispersi"e !echanismCopyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.

Second round

of replication

/irst round

of replication

Ori&inal

dou-le heli6

0c Dispersi"e mechanism( DNA replication produces DNA

strands in which se&ments of new DNA are interspersed

with the parental DNA(


34/70

34


Second round

of replication

/irst round

of replication

Ori&inal

dou-le heli6

8arental strand

Dau&hter strand

0a Semiconser"ati"e mechanism( DNA replication produces

DNA molecules with 1 parental strand and 1 newl% made

dau&hter strand(

0- Conser"ati"e mechanism( DNA replication produces 1 dou-le

heli6 with -oth parental strands and the other with ) new

dau&hter strands(

0c Dispersi"e mechanism( DNA replication produces DNA

strands in which se&ments of new DNA are interspersedwith the parental DNA(


35/70

In 16@2, !atthew !eselson and /ran$lin Stahl

de(ised an e%periment to dierentiate among the three

proposed >?= replication mechanisms

?itrogen comes in a common li&ht form )18

?* anda rare hea"% form )1@?*

Grew E. coli in medium with 1@? to la"el, then switched

to medium with 18?, collecting samples ater each

generation 'riginal parental strands would -e 1=N while newly

made strands would "e 18?

Conclusion& Semiconser"ati"e DNA replication35

!eselson and Stahl e6periment


36/70

36


© Meselson, M., !tahl, /., )16@2* The replication o >?= in Escherichia coli ,

P?=!, 88)J*&FJ1427, /ig. 8a

=

Appro6imate &enerations after transfer to 1N medium(

Li&ht

5alf4hea"%

5ea"%

1( *(1( )(

+5# DA+A1

*

)row -acteria in 1=Nmedia(

+ransfer to 1N media and

continue &rowth for 1,

1(, )(, or * &enerations(

1N medium

0li&ht

1=N medium

0hea"%

Isolate DNA after each &eneration( +ransfer

DNA to CsCl &radient, and centrifu&e(

DNA

CsCl &radient

Centrifu&e

O-ser"e DNA under 7V li&ht(


37/70

Semiconser"ati"e replication

The two parental strands separate and ser(e

as template strands

?ew nucleotides must o"ey the A+JC rule

And result& two new dou"le helices with same

"ase sequence as original

37


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38


+ A

+ A

AC

C

+ A

C

C

+ A

+ A

C

C

A

C

C

A +

A +

+ A

+ A

+ A

C

C

C

C

C C

C

A +

A +

A +

+ A

+ A

+ A

C

C

C

C

C

C

C

A +

A +

A +

+ A

+ A

+ A

C

C

C

C

C

C

C

A +

Incomin&

nucleotides

Ori&inal

0template

strand

Newl%

s%nthesi:ed

dau&hter strand

Ori&inal

0template

strand =E =E *E *E 0- +he products of replication0a +he mechanism of DNA replication

*E

=E *E =E *E

=E *E

*E =E

*E =E

A +

C

C

C

A +

C

+ A

=E

+ A

A +

A

C

+

+

Replication

for$A +

C

C


39/70

Ori&in of replication pro(ides an opening

called a replication "u""le that orms two

replication or+s

>?= replication proceeds outward rom or+s

;acteria ha(e sin&le ori&in o replication

Au+aryotes ha(e multiple ori&ins o replication

39

!olecular !echanism

of DNA Replication


40/70

40


)

1

*

)

DNA strands unwind(

DNA replication -e&ins outward

from two replication for$s(

DNA replication

continues in -oth

directions(

)

Replication

for$s

Replication

for$

Replication

for$

DNA replication

is completed(

Site where

DNA replication

endsDNA strands unwind,

and DNA replication

-e&ins(

DNA strands unwind,

and DNA replication

-e&ins at multiple

ori&ins of replication(

DNA replication

is completed(


41/70

DNA helicase

;inds to >?= and tra(els @ to using =TP to separate strand and mo(e or+

orward

DNA topoisomerase

5eli(es additional coiling ahead o

replication or+

Sin&le4strand -indin& proteins

eep parental strands open to act as

templates

41


42/70

42


=E

*E

*E

DNA topoisomerase

tra"els sli&htl% aheadof the replication for$

and alle"iates coilin&

caused -% the action

of helicase(

=E

DNA helicase tra"els

alon& one DNA strand

in the =E to *E direction

and separates the DNAstrands(

Direction of replication for$

Sin&le4strand -indin& proteins

coat the DNA strands to pre"ent

them from re4formin& a dou-le heli6(


43/70

DNA pol%merase

Co(alently lin+s nucleotides>eo%ynucleoside triphosphates

43


0a Action of DNA pol%merase

=E

*E Incomin&

deo6%nucleoside

triphosphates

+ e mp la t e s t rand

DNA pol%merase

catal%tic site


44/70

Deo6%nucleoside triphosphates /ree nucleotides with three phosphate groups

;rea+ing co(alent "ond to release pyrophosphate )twophosphates* pro(ides energy to connect nucleotidesCopyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.

55

5

55

OOO

O @

8N

O

5

N

O

C5*

O @

O

N

5N

N

N

5

N

5 5

5

55

OOO

O @

8C5)

O

O

5N

N

N5

N

5 5

5

55

OOO

O @

8C5)

O @

N

5

5

5

5 N

N

5

5

5

5

N

N

5 5

5

55

OOO

O @

8C5)

9

O

55

N

N

55

5

55

OO

O

8

O @

NO

O

5 5

N

N

5 5

5 O 5

O5

5 5

O O

O

8 C 5 ) O

@ O

O

8 O

O

8

@ O

O

8

O @

O

55

5

55

OOO

O @

8N

O 5

5

N

O

C5*

O

5N

N

N

5

N

5 5

5

55

OOO

O @

8

O

O

5N

N

N

5

N

5 5

5

55

OOO

O @

8C5)

O

@

5 N

N

5

N

N

5 5

5

55

OOO

O @

8C5)

O

55

N

N

55

5

55

OO

O

8 C5)

O @O

O

55

N

N

55

5

55

OO

O

8 C5)

O @O

O

5

+A

C

C

O @

@ O

O

8

O @

O

+

C

C

A

+

C

C

A

N

5

5

5

5 N

N

5

5

N

5

5

5

5N

N

5

5

5

5N

O O @ @O

O O

8 8

@O O @

=E end

C5)

C5)

*E end

5O

=E end

O @

C5)

+emplate

strand

*E end *E end

=E end

8hosphate

*E end 8%rophosphate

New phosphoester

-ond

=E end

C5)

O5 9

An incomin& nucleotide

0a deo6%nucleoside triphosphate

0- Chemistr% of DNA replication

O @ O

@ O

@

5O


45/70

/eatures of DNA pol%merase

1. >?= polymerase cannot -e&in s%nthesis

on a "are template strand

5equires a primer to get started >?= primase ma+es the primer rom 5?=

The 5?= primer is remo(ed and replaced with

>?= later

7. >?= polymerase onl% wor$s =. to *.

45


46/70

46

=E *E

*E =E

DNA pol%merase can lin$

nucleotides onl% in the=E to *E direction(

DNA pol%merase is a-le to

co"alentl% lin$ nucleotidesto&ether from a primer, which

is made -% DNA primase(

0- =E to *E direction of

DNA s%nthesis

0a Need for a primer

RNA

primer



47/70

Leadin& strand

>?= synthesiDed in as one lon& molecule>?= primase ma+es a single 5?= primer

>?= polymerase adds nucleotides in a @ to

direction as it slides orward

La&&in& strand

>?= synthesiDed @ to "ut as O$a:a$i fra&ments

'+aDa+i ragments consist o 5?= primers plus >?=

In -oth strands5?= primers are remo(ed "y >?= polymerase and

replaced with >?=

>?= ligase :oins ad:acent >?= ragments47


48/70

48


1

)

*

=E

=E

=E

*E

=E

*E

*E

*E

=E

=E

*E

*E

*E

DNA strands separate at an

ori&in of replication, creatin&

) replication for$s(

Replication

for$s

RNA primer Leadin&strand

8rimers are needed to initiate

DNA s%nthesis( +he s%nthesis

of the leadin& strand -e&ins in

the direction of the replication

for$( In the la&&in& strand, the

first O$a:a$i fra&ment is made

in the opposite direction(

+he leadin& strand elon&ates,and a second O$a:a$i fra&ment

is made(

=E

*E

=E

8rimer

/irst O$a:a$i fra&mentof the la&&in& strand

Direction of

replication for$

/irst

O$a:a$i

fra&ment

Second

O$a:a$i

fra&ment


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49


*E

=E

=E

*E

=E

=E

*E

*E

+he leadin& strand continues

to elon&ate( A third O$a:a$i

fra&ment is made, and the first

and second are connected

to&ether(

/irst and second O$a:a$i

fra&ments ha"e -een

connected to each other(

+hird

O$a:a$i

fra&ment


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50


=E

*E =E

*E

Leadin&

strandLa&&in&

strand

Replication

for$

Replication

for$

0- Replication from an ori&in

Ori&in of replication

Leadin&

strand

La&&in&

strand


51/70


)

*E

=E

=E

*E

=E

*E

=E

*E

=E

1DNA

primaseDNA primase ma$es RNA primers to -e&in

the replication process(

DNA pol%merase III ma$es DNA from the

RNA primers( DNA primase hops -ac$ to

the openin& of the for$ and ma$es a second

RNA primer for the la&&in& strand(

Direction of replication for$

DNA

pol%merase III

Second

primer

DNAprimase

/irst

RNA primer

DNA

pol%merase III

Leadin&

strand

Clamp

protein

RNA

primer

La&&in& strand

0O$a:a$i

fra&ment

*E

=E

*E

=E

*E

=E

*E

=E

*E

=E

*E

=E

* DNA pol%merase III continues to elon&ate

the leadin& strand( In the la&&in& strand,

DNA pol%merase III s%nthesi:es DNA

from the second primer( DNA pol%merase

I remo"es the first primer and replaces it

with DNA(

+hird

primer

Second

primer

!issin&

co"alent -ond

DNA li&ase

+hird

primer

In the la&&in& strand, DNA li&ase forms a

co"alent -ond -etween the first and second

O$a:a$i fra&ments( A third O$a:a$i

fra&ment is made( +he leadin& strand

continues to elon&ate(

DNA

pol%merase I

*E

=E

5eplicaciKn en E. coli


52/70

52

8lease note that due to differin&

operatin& s%stems, some animations

will not appear until the presentation is

"iewed in 8resentation !ode 0Slide

Show "iew( ;ou ma% see -lan$ slides

in the MNormal or MSlide Sorter "iews(

All animations will appear after "iewin&

in 8resentation !ode and pla%in& each

animation( !ost animations will reuire

the latest "ersion of the /lash 8la%er,

which is a"aila-le at

http2JJ&et(ado-e(comJflashpla%er(


53/70

DNA replication is "er% accurate

Three mechanisms or accuracy

1( 5%dro&en -ondin& "etween = and T,

and "etween G and C is more sta"le than

mismatched com"inations

7. =cti(e site o DNA pol%merase is unli+ely to orm

"onds i pairs mismatched

. >?= polymerase can proofread to remo(emismatched pairs

>?= polymerase "ac+s up and digests lin+ages

'ther >?= repair enDymes as well

53


54/70

DNA 8ol%merases Are a /amil% of

#n:%mes Gith Speciali:ed /unctions

Important issues or >?= polymerase are speed,

fidelit%, and completeness

?early all li(ing species ha(e more than one t%pe o>?= polymerase

Genomes o most species ha(e se(eral >?= polymerase

genes due to gene duplication

Independent genetic changes produce enDymes with

speciali:ed functions


55/70

E. coli has @ >?= polymerasesDNA pol%merase III 4 multiple su"units,

responsi"le or ma:ority o replication

DNA pol%merase I 4 a single su"unit, rapidly

remo(es 5?= primers and ills in >?=DNA pol%merases II, IV and V 4 >?= repair and

can replicate damaged >?=

>?= polymerases I and III stall at >?= damage

>?= polymerases II, IL and L dont stall "ut go slower

and ma+e sure replication is complete


56/70

Humans ha(e 17 or more >?= polymerases

>esignated with Gree+ letters

DNA pol%merase P @ its own "uilt in primase su"unit

DNA pol%merase Q and @ e%tend >?= at a aster rate

DNA pol%merase @ replicates mitochondrial >?=

hen >?= polymerases , N or O encounter a"normalities they

may "e una"le to replicate

0esion-replicating polymerases may "e a"le to synthesiDe

complementary strands to the damaged area


57/70


58/70

+elomeres

!eries o short nucleotide sequences repeated

at the ends of chromosomes in eu+aryotes

!pecialiDed orm o >?= replication only ineu+aryotes in the telomeres

Telomere at does not ha(e a complementary

strand and is called a *. o"erhan&

58


59/70

59


+ + A + + A + + A + + A

C C C A A + C C C A A + C C C A A +

+ + A

*E

=E

+elomere repeat seuences

*E o"erhan&


60/70

>?= polymerase cannot copy the tip o the

strand with a end

?o place or upstream primer to "e made

I this replication pro"lem were not sol(ed,

linear chromosomes would "ecome

pro&ressi"el% shorter

+elomerase en:%me attaches many copies

o >?= repeat sequence to the ends ochromosomes

60


61/70

!hortening o telomeres is correlated with

cellular senescence

Telomerase unction is reduced as an

organism ages

66 o all types o human cancers ha(e

hi&h le"els of telomerase

61


62/70


A

7C C AAC

=E

*E

*E

*E

=E

+elomere#u$ar%otic

chromosome

+elomere

RNA in

telomerase

+elomerase+elomerase s%nthesi:es

a 4nucleotide repeat

seuence(

+elomerase -inds to a

DNA repeat seuence(

1

)

+ A A + C C C A A + C C C A A + C C CA A 7 C C C

+ + A + + A + + AA A 7+ + A

C C C+ + A

A+

A A AA7 7C C CA + + A + + A + + A + + A + +

A + C C C A A +

=E

*E =E

*E

8rimase ma$es an RNA primer near the end of

the telomere, and DNA pol%merase s%nthesi:es

a complementar% strand in the =E to *E direction(+he RNA primer is e"entuall% remo"ed(

+elomerase mo"es nucleotides

to the ri&ht and -e&ins to ma$e

another repeat(

RNA primer that ise"entuall% remo"ed

*

+ A + + A + + A + + AA A 7

A + C C C A A +

=E

=E

*E

Repeat seuence

A + + A + + A + +++

A A A 7 C C C A A 7

+A + C C C A A +


63/70

Typical eu+aryotic chromosome may "e

hundreds o millions o "ase pairs long

0ength would "e 1 meter ;ut must it in cell 13-133Qm

Chromosome

>iscrete unit o genetic material

Chromosomes composed o chromatin

>?=-protein comple%

63

!olecular Structure of

#u$ar%otic Chromosomes


64/70

+hree le"els of DNA compaction

1( DNA wrappin&

>?= wrapped around histones to orm nucleosome

!hortens length o >?= molecule J-old

)( *4nm fi-er

Current model suggests asymmetric, > DigDag o

nucleosomes

!hortens length another J-old

64


65/70

65


Nucleosome2 histone proteins 9

1 or 1? nucleotide

-ase pairs of DNA

DNA

Lin$er

re&ion

Amino

terminal

tail of

histoneprotein

5)B5)B

55

5)A

5*

51

11 nm

* nm

0a !icro&raph of a *4nm fi-er

0- +hree4dimensional :i&:a& model

a& Photo courtesy o >r. ;ar"ara Ham+aloR



66/70

*( Radial loop domains

Interaction "etween

3-nm i"ers and

nuclear matri%

Aach chromosome

located in discreteterritory

0e(el o compaction is

not uniorm

5eterochromatin

#uchromatin

66


e n e

e n e

e n

e

8rotein fi-er inside the nucleus

*4nm fi-er

Radial loop

domain

8rotein that attaches the -ase

of a DNA loop to a protein fi-er


67/70

Cell di"ision

hen cells prepare to di(ide, chromosomes

"ecome e(en more compacted

#uchromatin not as compact

5etrochromatin much more compact

Metaphase chromosomes highly compacted

67


68/70

68


) nm

* nm

1

)

11 nm

5istone 51

Grappin& of DNA around

histone proteins

/ormation of a *4dimensional

:i&:a& structure "ia histone

51 and other DNA4-indin&

proteins

5istones

Nucleosome

DNA dou-le heli6

0a DNA dou-le heli6

0- Nucleosomes 0M-eads on a strin&

0c *4nm fi-er

a& © >r. Gopal Murti$Lisuals #nlimited9 "& © =da 0. 'lins and >onald A. 'lins$;iological Photo !er(ice9 c& Courtesy >r. Eerome ;. 5attner,

Cell ;iology and =natomy, #ni(ersity o Calgary


69/70

69


*

=

Anchorin& of radial loop

domains to the nuclear matri6

/urther compaction of radial

loops to form heterochromatin

!etaphase chromosome with

) copies of the DNA

1, nm

? nm

* nm

0d Radial loop domains

0e 5eterochromatin

0f !etaphase chromosome

d& Courtesy o Paulson, E.5. S 0aemmli, #.. Eames 5. Paulson, #.. 0aemmli, The structure o histonedepleted

metaphase chromosomes, Cell , 17&21J472, Copyright Alse(ier 16JJ9 e-& © Peter Angelhardt$

>epartment o Lirology, Haartman Institute



70/70

) nm

* nm

1

*

)

=

11 nm

5istone 51

Grappin& of DNA around

histone proteins

/ormation of a *4dimensional

:i&:a& structure "ia histone

51 and other DNA4-indin&

proteins

Anchorin& of radial loop

domains to the nuclear matri6

/urther compaction of radial

loops to form heterochromatin

!etaphase chromosome with

) copies of the DNA

1, nm

? nm

* nm

5istones

Nucleosome

DNA dou-le heli6

0a DNA dou-le heli6

0- Nucleosomes 0M-eads on a strin&

0c *4nm fi-er

0d Radial loop domains

0e 5eterochromatin

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Cap 11 biologia