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1
Chapter 11
Lecture and
Animation Outline
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!ee separate PowerPoint slides or all igures and
ta"les pre-inserted into PowerPoint without notes and
animations.
To run the animations you must "e in Slideshow View. #se
the "uttons on the animation to play, pause, and turn
audio$te%t on or o.
Please Note& 'nce you ha(e used any o the animation
unctions )such as Play or Pause*, you must irst clic+ on the
slides "ac+ground "eore you can ad(ance to the ne%t slide.
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Chapter 11
Nucleic Acid Structure, DNA Replication,
and Chromosome Structure
Biochemical Identification of the enetic !aterial
Nucleic Acid Structure
An O"er"iew of DNA Replication
!olecular !echanism of DNA Replication
!olecular Structure of #u$ar%otic Chromosomes
2
ey Concepts&
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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
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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
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5
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
+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(
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!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
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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
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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
)
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
#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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Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
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
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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
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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.
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12
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
* 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(
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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
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14
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
Sin&le strand
Nucleotides
Dou-le heli6
DNA associates with
proteins to form a
chromosome(
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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
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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*
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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
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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
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!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
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
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
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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
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
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 @
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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$lin.s
H4ra% diffraction results
were crucial e(idence,
suggesting a helical structure
with uniorm diameter
21
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22
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
H4ra%s diffracted -% DNA
onto photo&raphic plate
8attern represents the
atomic arra% in wet fi-ers
Get DNA fi-ers
H4ra% -eam
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#rwin Char&off analyDed "ase composition
o >?= rom many dierent species
5esults consistently showed
amount o adenine )=* B amount o thymine )T*
amount o cytosine )C* B amount o guanine )G*
23
Base4pairin&
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24
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Gatson and Cric$
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
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Dou-le stranded
Antiparallel strands
Ri&ht4handed heli6
Su&ar4phosphate
-ac$-one
Bases on the inside
!ta"iliDed "y 54-ondin&
!peciic -ase4pairin&
13 nts per helical turn 26
/eatures of DNA
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
Bases
5%dro&en -ond
) nm
0a Dou-le heli6
=E end
*E end
Su&ar4phosphate
-ac$-one
One nucleotide
(* nm
*E end
=E end
Complete turn
of the heli6
*( nm
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Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
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&
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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
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Groo(es are re(ealed in
the space-illing model
!a3or &roo"e
Proteins "ind to aect genee%pression
!inor &roo"e
?arrower
29
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
!a3or &roo"e
!inor &roo"e
!a3or &roo"e
!inor &roo"e
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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
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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(
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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(
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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(
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34
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
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(
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In 16@2, !atthew !eselson and /ran$lin Stahl
de(ised an e%periment to dierentiate among the three
proposed >?= replication mechanisms
?itrogen comes in a common li&ht form )18
?* anda rare hea"% form )1@?*
Grew E. coli in medium with 1@? to la"el, then switched
to medium with 18?, collecting samples ater each
generation 'riginal parental strands would -e 1=N while newly
made strands would "e 18?
Conclusion& Semiconser"ati"e DNA replication35
!eselson and Stahl e6periment
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36
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
© Meselson, M., !tahl, /., )16@2* The replication o >?= in Escherichia coli ,
P?=!, 88)J*&FJ1427, /ig. 8a
=
Appro6imate &enerations after transfer to 1N medium(
Li&ht
5alf4hea"%
5ea"%
1( *(1( )(
+5# DA+A1
*
)row -acteria in 1=Nmedia(
+ransfer to 1N media and
continue &rowth for 1,
1(, )(, or * &enerations(
1N medium
0li&ht
1=N medium
0hea"%
Isolate DNA after each &eneration( +ransfer
DNA to CsCl &radient, and centrifu&e(
DNA
CsCl &radient
Centrifu&e
O-ser"e DNA under 7V li&ht(
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Semiconser"ati"e replication
The two parental strands separate and ser(e
as template strands
?ew nucleotides must o"ey the A+JC rule
And result& two new dou"le helices with same
"ase sequence as original
37
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38
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
+ A
+ A
AC
C
+ A
C
C
+ A
+ A
C
C
A
C
C
A +
A +
+ A
+ A
+ A
C
C
C
C
C C
C
A +
A +
A +
+ A
+ A
+ A
C
C
C
C
C
C
C
A +
A +
A +
+ A
+ A
+ A
C
C
C
C
C
C
C
A +
Incomin&
nucleotides
Ori&inal
0template
strand
Newl%
s%nthesi:ed
dau&hter strand
Ori&inal
0template
strand =E =E *E *E 0- +he products of replication0a +he mechanism of DNA replication
*E
=E *E =E *E
=E *E
*E =E
*E =E
A +
C
C
C
A +
C
+ A
=E
+ A
A +
A
C
+
+
Replication
for$A +
C
C
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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
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40
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
)
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(
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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
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42
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
=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(
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DNA pol%merase
Co(alently lin+s nucleotides>eo%ynucleoside triphosphates
43
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
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
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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
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/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$s =. to *.
45
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46
=E *E
*E =E
DNA pol%merase can lin$
nucleotides onl% in the=E to *E direction(
DNA pol%merase is a-le to
co"alentl% lin$ nucleotidestoðer from a primer, which
is made -% DNA primase(
0- =E to *E direction of
DNA s%nthesis
0a Need for a primer
RNA
primer
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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
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48
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
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
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
*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
toðer(
/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
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
=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
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Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
)
*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
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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(
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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
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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
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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
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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
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+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
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59
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
+ + 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&
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>?= 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
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!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
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Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
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$e
another repeat(
RNA primer that ise"entuall% remo"ed
*
+ A + + A + + A + + AA A 7
A + C C C A A +
=E
=E
*E
Repeat seuence
A + + A + + A + +++
A A A 7 C C C A A 7
+A + C C C A A +
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Typical eu+aryotic chromosome may "e
hundreds o millions o "ase pairs long
0ength would "e 1 meter ;ut must it in cell 13-133Qm
Chromosome
>iscrete unit o genetic material
Chromosomes composed o chromatin
>?=-protein comple%
63
!olecular Structure of
#u$ar%otic Chromosomes
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+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
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65
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
Nucleosome2 histone proteins 9
1 or 1? nucleotide
-ase pairs of DNA
DNA
Lin$er
re&ion
Amino
terminal
tail of
histoneprotein
5)B5)B
55
5)A
5*
51
11 nm
* nm
0a !icro&raph of a *4nm fi-er
0- +hree4dimensional :i&:a& model
a& Photo courtesy o >r. ;ar"ara Ham+aloR
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
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*( Radial loop domains
Interaction "etween
3-nm i"ers and
nuclear matri%
Aach chromosome
located in discreteterritory
0e(el o compaction is
not uniorm
5eterochromatin
#uchromatin
66
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
e n e
e n e
e n
e
8rotein fi-er inside the nucleus
*4nm fi-er
Radial loop
domain
8rotein that attaches the -ase
of a DNA loop to a protein fi-er
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Cell di"ision
hen cells prepare to di(ide, chromosomes
"ecome e(en more compacted
#uchromatin not as compact
5etrochromatin much more compact
Metaphase chromosomes highly compacted
67
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68
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
) nm
* nm
1
)
11 nm
5istone 51
Grappin& of DNA around
histone proteins
/ormation of a *4dimensional
:i&:a& structure "ia histone
51 and other DNA4-indin&
proteins
5istones
Nucleosome
DNA dou-le heli6
0a DNA dou-le heli6
0- Nucleosomes 0M-eads on a strin&
0c *4nm fi-er
a& © >r. Gopal Murti$Lisuals #nlimited9 "& © =da 0. 'lins and >onald A. 'lins$;iological Photo !er(ice9 c& Courtesy >r. Eerome ;. 5attner,
Cell ;iology and =natomy, #ni(ersity o Calgary
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69
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
*
=
Anchorin& of radial loop
domains to the nuclear matri6
/urther compaction of radial
loops to form heterochromatin
!etaphase chromosome with
) copies of the DNA
1, nm
? nm
* nm
0d Radial loop domains
0e 5eterochromatin
0f !etaphase chromosome
d& Courtesy o Paulson, E.5. S 0aemmli, #.. Eames 5. Paulson, #.. 0aemmli, The structure o histonedepleted
metaphase chromosomes, Cell , 17&21J472, Copyright Alse(ier 16JJ9 e-& © Peter Angelhardt$
>epartment o Lirology, Haartman Institute
Copyright © The McGraw-Hill Companies, Inc. Permission required or reproduction or display.
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) 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