✅L4, 5 and 6 DNA synthesis(1).pdf files.

It's for studying dna

1. DNA SYNTHESIS

2. Objectives
•DefineDNAreplication
•RecognizedifferentmodelsofDNAreplication
(semiconservative,conservativeanddispersive)
•Describethereplicationfork
•ListthecomponentsrequiredtoinitiateDNAreplication
•ExplainthestepsofDNAreplication
•Differentiateleadingandlaggingstrand
References:
Lippincott'sIllustratedReviewsofBiochemistry,5thEdition,
UnitVI,chapter29(DNAstructure,Replicationand
Repair)

3. Objectives
§Statetheroleofdifferentenzymesusedduringthe
DNAreplication
§Understandthevarioustypesofnucleasesthat
affectnucleicacidstructure
§ClassifyprokaryoticandeukaryoticDNA
polymerases
§Definetelomeresandtelomeraseexpresstheirrole
inDNAstability
References:
Lippincott'sIllustratedReviewsofBiochemistry,5th
Edition,UnitVI,chapter29(DNAstructure,Replication
andRepair)

4. §DNA replicationistheprocessby
whichDNAmakesacopyofitself.
§DNA replicationinprokaryotesand
eukaryotesoccursbeforethedivisionofcells.
§DNAsynthesis(replication)ineukaryotesoccurs
duringthe(Sphase)ofcellcyclebeforecell
division.DNAreplicationoccursoncepercell
cycle.
§Whenthecelldivides(Mitosis),eachoneofthe
daughtercellswillhaveacopyfromthese
chromosomes(DNA).
DNA
1.Sm
chromatinb
chromosome
nucleosome
DNA
+
Histone
23Miosis
-23
=>
46 youIn'tmake
them
duringstarvation
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methotroxateinhibst
Folic
acid
formation

5. bysignals
(hormone)
=
3,4545

6. §Themajorenzymaticfunctionscarriedoutatthe
processofDNAreplicationarewellconserved
fromprokaryotestoeukaryotes,butthe
replicationmachineryineukaryoticDNA
replicationisamuchlargercomplex.
§DNA replicationwasprovedtobe
semiconservative.

7. Models of DNA replication
threemodelsofreplicationpossiblefromsuchascheme:
conservative,semi-conservative,anddispersive.
Inconservativereplication:
ThetwooriginalDNAstrands(oldstrand,knownasthe
parentalstrands)wouldre-basepairwitheachotherafter
beingusedastemplatestosynthesizenewstrands;and
thetwonewly-synthesizedstrands,knownasthe
daughterorcomplementarystrands,wouldalso
basepairwitheachother;
oneofthetwoDNAmoleculesafterreplicationwouldbe
“all-old”andtheotherwouldbe“all-new”.
distribute
six Six s?.DNA" s

8. Models of DNA replication
Insemi-conservativereplication:
EachofthetwoparentalDNAstrandswouldactasa
templatefornewDNAstrandstobesynthesized,butafter
replication,eachparentalDNAstrandwouldbasepair
withthecomplementarynewly-synthesizedstrandjust
synthesized,andbothdouble-strandedDNAswould
includeoneparentalor“old”strandandonedaughteror
“new”strand.
Indispersivereplication:
AfterreplicationbothcopiesofthenewDNAswould
somehowhavealternatingsegmentsofparentalDNA
andnewly-synthesizedDNAoneachoftheirtwostrands.

9. -
a
is
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--
-
s6So
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atthe
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10. we
ParentStrands
(3,5
I se
or
tempelates

11. Steps of replication
Step1:Initiation:
A.ReplicationForkFormation.BeforeDNA
canbereplicated,thedoublestranded
moleculemustbe“unzipped”intotwosingle
strands.
B.PrimerBinding.Theleadingstrandisthe
simplesttoreplicate.
Step2:Elongation.
Step3:Termination.

12. INITIATION

13. DnaAprotein
•DnaAproteinbindstospecificnucleotide
sequencesattheoriginofreplication(ori),
causingshort,tandemlyarranged(oneafterthe
other)AT-richregionsintheorigintomelt.
•MeltingisATP-dependent,andresultsin
strandseparationwiththeformationof
localizedregionsofssDNA.
easier
G
At
I
bonds
GC
3
bonds

14. ONAAIs
replicationbubble
template
bubble
is
isreplications
new
5
-
-
3
/3
-
5Replication
lagging
strand fork replication
discontineous fork
latent

15. Pro
EU
circularDNA
ori
-IDNASi
⑤81
-
-
separation
+
replicationss'$
-" :
6
Pro
replicationbubble
"¥is:
S
!3.1 t
:05/1
↑
easierseparationA =T
=
22
bond's
0'S
c
=
6
-
x3
bonds

16. Components of replication fork
1.ProteinsrequiredforDNAstrand
separation:
a.DnaAprotein
b.Helicasesenzyme
c.Single-strandedDNA-bindingproteins
(SSB)
2.Primase(aspecificRNApolymerase)
replications
i'sdo'
ori,
I'
5
:
:25
-
separating
bubbles,
-; 5
unwinding
theDNA
unzipping
melting
·
See Ses.
&
protectthe
singlestrandfromnucleaseenzyme·
-
1
RNA
primer
I
RNAneoclutide
~
aPrime
jei'?
?
I
Gas:
--
⑤is
'
-Elongations.Is
Is

17. DNA helicases
•TheseenzymesbindtossDNAnearthe
replicationfork,andthenmoveintothe
neighboringdoublestrandedregion,forcing
thestrandsapartineffect,unwinding
(unzipping)thedoublehelix.
•HelicasesrequireenergyprovidedbyATP.
•Note:DnaBistheprincipalhelicaseof
replicationinE.coli.

19. Single-stranded DNA-binding (SSB)
proteins
•TheseproteinsbindtothessDNAgeneratedby
helicases.
•TheSSBproteinsarenotenzymes,These
proteinsnotonlykeepthetwostrandsofDNA
separatedintheareaofthereplicationorigin,
thusprovidingthesingle-strandedtemplate
requiredbypolymerases,butalsoprotectthe
DNAfromnucleasesthatdegradessDNA.
G

20. daughter
-
new-complementry
old-pavent
-
template

21. Primase
•AspecificRNApolymerase,calledprimase(DnaG),synthesizes
theshortstretchesofRNA(approximatelytennucleotideslong
calledprimers)thatarecomplementaryandantiparalleltothe
DNAtemplate.
•Intheresultinghybridduplex,theUinRNApairswithAinDNA.
•Theseprimersareconstantlybeingsynthesizedatthereplication
forkonthelaggingstrand(multipleprimers),butonlyoneRNA
sequenceattheoriginofreplicationisrequiredontheleading
strand.
•Thesubstratesforthisprocessare5'-ribonucleoside
triphosphates,andpyrophosphateisreleasedaseach
ribonucleosidemonophosphateisaddedthroughformationofa
3'→5'phosphodiesterbond.
•TheRNAprimerislaterremoved.
MixDNA,RNA
W
2
phosphat

23. Replication fork
Esse
ori
~
Polaritys'igs
-
3
-
-
9
I
-
5
-
- 3

24. ELONGATION

25. I
have
primer:
byRNApolymerase
-
2direction
from
5'--5'
atthe
new
strand
Phosphodiester
SSNucleotide
3
-
&-

26. at
new
strand5'-3'
template
new
has
a
polarity'-s8'I
-
new
3
-
-
5
withmultipleprimers
SNAPolymerase
&
-
DNApolymerase
S.j s
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Polymerase
5'-3'
-01.s,1,
multipleprimers'-48is,er
5,5.s,
I,
Ss,
-
⑱

27. new
strand.
leadingstrandtemplate
3
-
x
5
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-
I
nucleono",diesterI
·ga:
-
·
Phosphodiester
-
->
$
DNApolymerasefunction
b
soit'sneedmultiple
primer
I-addnew
nucleotide-- 3
andthe
new
DNA
willbe 2.
Proofreading
!Eis
a
multiplefragments
->- : 2,ost55
phosphodiestersists:dexonuclease8S.-5.S
bond
3-5exonuclease
activity's
I'
S
template
from5-3
so
the
new
willbe
3'-ss(lagging
strand)

28. DNAPol
I
9'sislending.
See?lagging&
- Y
is
DNA-S. INA
ISs'e'-fragmentSc
- is sessive
primer-
S'-3'exonuclease
activity
DNA isRNA"50Polymeraseactivity
-
2
&
:
,
I9056Ssa DNA
Is S-3'-s'proof
readingexonuclease
activity
-3
-
1,
%.
-
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DNAligase 3,
-
okaza
11
~ . . .
Ga~Is
5.5-
s
framentI
function:sealing
ofphosphodiesterbonk
b
⑨
between
primer
end
+
okazakistart
needs
2ATP
6
Phosphodiester
-
gap:? Nudertide
is:"I do
bond

29. DNA polymerase III
•DNAchainelongationiscatalyzedbyDNApolymeraseIII.
•Usingthe3'-hydroxylgroupoftheRNAprimerastheacceptorof
thefirstdeoxyribonucleotide,DNApolymeraseIIIbeginstoadd
nucleotidesalongthesingle-strandedtemplatethatspecifiesthe
sequenceofbasesinthenewlysynthesizedchain.
•DNApolymeraseIIIisahighly“processive”enzyme—thatis,it
remainsboundtothetemplatestrandasitmovesalong,anddoesnot
diffuseawayandthenrebindbeforeaddingeachnewnucleotide.
•TheprocessivityofDNApolymeraseIIIistheresultofitsβ
subunitformingaringthatencirclesandmovesalongthetemplate
strandoftheDNA,thusservingasaslidingDNAclamp.
•Thenewstrandgrowsinthe5'→3'direction,antiparalleltothe
parentalstrand.
-
"seeds;s
&I
50
nucleotide/s
& -"sigis rs's~is
:=
-SONASS-
/
-5.
1
Polymerisation&
is
5
2
-
proofreading

30. DNA polymerase III
•Thenucleotidesubstratesare5'-deoxy-ribonucleoside
triphosphates.Pyrophosphate(PPi)isreleasedwheneachnew
deoxynucleosidemonophosphateisaddedtothegrowingchain.
•HydrolysisofPPito2Pimeansthatatotaloftwohigh-energy
bondsareusedtodrivetheadditionofeachdeoxynucleotide.
•Allfourdeoxyribonucleosidetriphosphates(dATP,dTTP,dCTP,
anddGTP)mustbepresentforDNAelongationtooccur.
•Ifoneofthefourisinshortsupply,DNAsynthesisstopswhen
thatnucleotideisdepleted.
leading$
- ~
strand
⑤s

31. -
⑪

32. Proofreading of newly synthesized
DNA
•DNApolymeraseIIIhas,inadditiontoits
5'→3'polymeraseactivity,a“proofreading”
activity3'→5'exonuclease.
•Theproofreadingexonucleaseactivityrequires
movementinthe3'→5'direction,not5'→3'
likethepolymeraseactivity.Thisisbecause
theexcisionmustbedoneinthereverse
directionfromthatofsynthesis
D
⑰

33. Proof
reading
&goa5 S-
-

34. -

35. 1
-
-
Primer
5j.
I,I
2
=
-
nucleotide's
-
directionat
new
strand5'-s3'
G?50 s
s
S
5
-
Primer
okazaky39,8

36. 1-
DNA
Polymerase
III
-2.
DNApolymerase
I
da
3-DNA
ligase
new
strandalwaysshorterthanoldd
->
Smallergenome
withmitosis
g.
,
.55
primeris;d=
3i0s:s
Polymerase;
-
*
is ses
we:
is i!
96
35:
:598

37. DNA ligase
•Thefinalphosphodiesterlinkagebetweenthe
5'-phosphategroupontheDNAchain
synthesizedbyDNApolymeraseIIIandthe3'-
hydroxylgrouponthechainmadebyDNA
polymeraseIiscatalyzedbyDNAligase.
•ThejoiningofthesetwostretchesofDNA
requiresenergy,whichinmostorganismsis
providedbythecleavageofATPtoAMP+
PPi.
③
=>
2A

39. Eukaryotic DNA Polymerases
prokaryots
enzymes
is
$5
multiplereplicationbubbleis
3.
SS5 esprimers
-
INApolymeraseinprokaryotes %95i
-
-
Ss
1i
·
S Proofreading
9
"9
accumulationofmutation
-
big
-
S
-
cancer
;
1499:645,6
--
-

40. TERMINATION

41. Termination of replication
•TerminationofDNAreplicationoccurswhen
tworeplicationforksmeetonthesamestretch
ofDNA.
•Anyremaininggapsarefilledandligated
(sealed).
•Replicationproteinsareunloaded.

42. -
- 199.555,0
&.
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replication
is
5-

43. replicationfork
21%

44. Enzymes solving the problem of
supercoils
~15.sli"- Sioss;d
"iis s

45. super
coil
-
-
-
i
-
&
o
poisomerase·S
-
coiling
ofcoil

46. What is DNA supercoil?
•“Supercoiling”meansthecoilingofacoil.
•DNAiscoiledintheformofadoublehelix,withboth
strandsoftheDNAcoilingaroundanaxis.Thefurther
coilingofthataxisuponitselfproducesDNA
supercoiling.
•DNAsupercoilingisgenerallyamanifestationof
structuralstrain.
•PositivesupercoilingofDNAoccurswhentheright-
handed,double-helicalconformationofDNAistwisted
eventighter(twistedinaright-handedfashion)untilthe
helixbeginstodistort.
•Negativesupercoiling,ontheotherhand,involves
twistingagainstthehelicalconformation(twistingina
left-handedfashion).
xi

47. Type I DNA topoisomerases
•Theseenzymesreversiblycutonestrandofthe
doublehelix.
•Theyhavebothnuclease(strand-cutting)and
ligase(strand-resealing)activities.
•TheydonotrequireATP,butratherappeartostore
theenergyfromthephosphodiesterbondthey
cleave,reusingtheenergytoresealthestrand.
•Eachtimeatransient“nick”iscreatedinoneDNA
strand,theintactDNAstrandispassedthroughthe
breakbeforeitisresealed,thusrelieving
(“relaxing”)accumulatedsupercoils.
•TypeItopoisomerasesrelaxsupercoilsinE.coliand
ineukaryoticcells.
&x;
is
ro'
Dir& sagosts.i
-
releaseenergy
4-
i)
-
sealing
-o use
thisenergy Care
Gragtecag
see
:
negative
Positive
ofnegative

48. Type II DNA topoisomerases
•TheseenzymesbindtightlytotheDNAdoublehelixandmake
transientbreaksinbothstrands.
•ThisisanATP-requiringprocess.
•TypeIIDNAtopoisomerasesarealsorequiredinboth
prokaryotesandeukaryotesfortheseparationofinterlocked
moleculesofDNAfollowingchromosomalreplication.
•DNAgyrase,aTypeIItopoisomerasefoundinbacteriaand
plants,hastheunusualpropertyofbeingabletointroduce
negativesupercoilsintorelaxedcircularDNAusingenergy
fromthehydrolysisofATP.Thisfacilitatesthefuturereplication
ofDNAbecausethenegativesupercoilsneutralizethepositive
supercoilsintroducedduringopeningofthedoublehelix.
sealing
&q
isigsIstrands
$
gri;514-4,

49. •Sometypesofanticanceragents,target
humantopoisomeraseII.
•BacterialDNAgyraseisauniquetargetofa
groupofantimicrobialagentscalled
quinolones,forexample,ciprofloxacin.
Q
type
oftopoisomerase
I
-
supercoil
-owillmake
-
supercoil
gyrase
isisasil'sis s 0b0s-digogads
die
&
supercoil-1 15,
9
"s95!
tive,!%813's:11
&
j
s
antibiotics!I

51. Telomere and Telomerase
•Telomeresarecomplexesofnon-codingDNAplus
proteinslocatedattheendsoflinearchromosomes.
•Theymaintainthestructuralintegrityofthe
chromosome,preventingattackbynucleases.
•Inhumans,itisformedofseveralthousandtandem
repeatsofanoncodinghexamericsequence,AG3T2,
base-pairedtoacomplementaryregionofCsandAs.
&S
::
-
RNA
template.RNA
ois
·70sgenow eve
seePoly
merase
reverse
transcription
reverse
transcription
*
S..INA=x
reverse
transcriptase[as
"
SDNA
&
s
repeated
sequence. S
.
S.-
:
oftelomer
G
&
"gos'ssitys'snoncodingPNAi
-
extension
of
genome
3.S
exonucleases"'s:"';'nucleases
i-Nga-DNA
-
,
3.
! ,extension:
-
telomeres
I
* &
mitosis's
is
is"s'sfolemere
6 y
-&
Cmitoticclock)
9
-
-
:,17,igd
:515

52. ↓
⑤
s
.
SSS
I
DNA
-
"'noncodingsos"".
' mitosis
:/
*
s
-
-
o< Sie!j0ig
multiplemitosis
->
shortergenome
--
aging
-inactivetelomerase
cancer
cell--reactivation
E
Cancer
gotoshort
tomez

54. •TheGT-richstrandislongerthanitsCA
complement,leavingssDNAafewhundred
nucleotidesinlengthatthe3'-end.
•Thesingle-strandedregionisthoughttofold
backonitself,formingaloopstructurethatis
stabilizedbyprotein.
telomeresshortenwitheachsuccessive
celldivision.Whenitisshortenedbeyond
somecriticallength,thecellisnolongerable
todivideandissaidtobesenescent.

55. •Itisacomplexenzymecontainsaproteinthat
actsasareversetranscriptase,andashortpiece
ofRNAthatactsasatemplate.
•ThereversetranscriptaseusestheRNAtemplate
tosynthesizetelomereDNA.

56. Inhibition of DNA synthesis by
nucleoside analogs
·
jeN
5s ->
drugtostop
&siis,zibars virus
replication
&
92',05
articancer
2 0
Eiso
-