Structure and Synthesis of DNA

This lecture note describes the structure of DNA, its component, forms of DNA and its replication process on prokaryotic and eukaryotic organisms.

1. Chapter Two
Synthesis and structure of DNA
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2. Chapter Objectives
After the end of this chapter the will able to
Define how cells synthesis nucleic acids
Mention the components of DNA
Explain the structure and functions of nucleic acid
Discuss different types of nucleic acids
Discuss the role of different enzymes in nucleic acid replication
Demonstrate the process of DNA replication
Define DNA mutation and repairing mechanisms
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3. Introduction
Nucleicacidsarebiopolymers/polynucleotides/composedofnucleotidesubunits
linkedbyphosphodiesterbonds.
Therearetwotypesofnucleicacids:deoxyribonucleicacid(DNA)andribonucleic
acid(RNA).
DNAencodeshereditaryinformation,controlscelldivision,growth&development.
Nucleotidetriphosphateservesassubstrateprecursorforbiosynthesisofnucleicacids.
Nucleotidesarelinkedbynucleophilicattackof3’-OHofonenucleotidetriphosphate
on5’phosphorusofanothernucleotide
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4. Con’t
backboneofalternatingphosphategroupseitherdeoxyriboseorribosepentosesugarjoinedby
phosphodiesterbond.
Heterocyclicbasesprimarilyadenine(A),guanine(G),cytosine(C),thymine(T)oruracil(U)arelinked
byhydrogenbonding.
ThesenitrogenousbasesarePurine&pyrimidines.
DNAhasdoubleintertwininghelicalstrandswithdeoxyribosesugarA,G,C,andTbases.
WhereasRNAissinglestrandedpolynucleotideswithribosesugar,A,G,C,andUheterocyclicbases.
ThebasesequencesofeachDNAstrandarecomplementarysothatbasepairsareA═TandG≡C.
BasepairinginRNAsoccursbetweenbasesonthesamefoldedstrand.
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5. DNA
What is DNA?
Isthegeneticmoleculescarryingallthegeneticinformationwithin
chromosome
Itiscomplexmoleculethatcontainsalloftheinformationnecessary
tobuildandmaintainanorganism.
DNAisamoleculethatcontainstheinstructionsanorganismneeds
todevelop,liveandreproduce.
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6. The components of nucleotides
The monomeric units for nucleic acids are
nucleotides
Nucleotides are made up of three structural subunits
Sugar: ribose in RNA, -deoxyribose in DNA
Heterocyclic base
Phosphate
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7. Nucleoside, nucleotides and nucleic acids
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The chemical linkage between monomer units
in nucleic acids is a phosphodiester bond.

8. DNA structure and properties
DNAisstableformofdoublehelixwith2distinctsizesofgrooves
majorgroove&minorgrooverunninginspiralfashion.
MostDNAproteinassociationsareinmajorgrooves.
Asinglestrandofnucleotideshasnohelicalstructure.
HelicalshapeofDNAdependsentirelyonthepairing&stackingof
thebasesintheantiparallelstrandsDNAhandedhelix.
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9. DNA structure and properties
PrimaryStructure:thebasesequenceor
nucleotidesequenceinpolydeoxynucleotide
chains
Itistheorderofbasesonthepolynucleotide
sequence;theorderofbasesspecifiesthegenetic
code.
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10. Secondary Structure
The3DconformationofthepolynucleotidesbackboneintodoublehelixofDNA.
Ittherelativespatialpositionofalltheatomsofnucleotideresidues
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11. DNA Double Helix & Hydrogen bonding
DNAismadeoftwopolynucleotidechains,wherethebackboneis
constitutedbysugar-phosphate,andthebasesprojectinside.
Thetwochainshaveanti-parallelpolarity.Itmeans,ifonechain
hasthepolarity5’3’,theotherhas3’5’.
Thebasesintwostrandsarepairedthroughhydrogenbond(H-
bonds)formingbasepairs(bp).
AdenineformstwohydrogenbondswithThyminefromopposite
strandandvice-versa.
Similarly,GuanineisbondedwithCytosinewiththreeH-bonds.
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12. BasedontheobservationofErwinChargaffthatforadoublestrandedDNA,theratiosbetweenAdenine
andThymine;andGuanineandCytosineareconstantandequalsone.
Hydrogenbond:-Achemicalbondconsistingofahydrogenatombetweentwoelectronegativeatoms
(e.g.,oxygenornitrogen)withonesidebeacovalentbondandtheotherbeinganionicbond.
12DNA Double Helix & Hydrogen bonding

13. Tertiary structures
ManynaturallyoccurringDNAmoleculesarecircular,withnofree5′or3′end.
DuetothepolarityofthestrandsoftheDNAdoublehelix,the5′endofonestrandcanonlyjoinitsown
3′endtocovalentlycloseacircle.
ThestructureofDNAdoesnotonlyexistassecondarystructuressuchasdoublehelices.
butitcanfolduponitselftoformtertiarystructuresbysupercoiling.
TertiarystructuresDNArefersthesupercoilingthe3Darrangementofallatomsofnucleicacid.
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14. DNA Supercoiling
DNA supercoiling refers to the over-or under-winding of a DNA strand, and is an expression of the
strain on that strand.
Supercoiling is important in a number of biological processes, such as compacting DNA.
Additionally, certain enzymes such as topoisomerases are able to change DNA topology to facilitate
functions such as DNA replication or transcription.
There are two types of DNA Super Coiling.
Positive DNA Supercoiling
Negative DNA supercoiling
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15. Positive DNA Supercoiling
Positivesupercoilingistheleft-handed,coilingofDNAthuswindingoccursinthe
clockwisedirection.
Thisprocessisalsoknownasthe"overwinding"ofDNA.
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16. Negative DNA supercoiling
Negative supercoiling is the right-handed coiling of DNA thus winding occurs in the
counterclockwise direction.
It is also known as the "underwinding" of DNA.
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17. Importance of DNA Supercoiling
DNA supercoiling is important for DNA packaging within all cells.
Because the length of DNA can be thousands of times that of a cell, packaging this genetic material into the cell or nucleus
(in eukaryotes ) is a difficult feat.
Supercoiling of DNA reduces the space and allows for much more DNA to be packaged.
In prokaryotes, plectonemicsupercoils are predominant, because of the circular chromosome and relatively small amount
of genetic material.
In eukaryotes, DNA supercoiling exists on many levels of both plectonemicand solenoidal supercoils, with the solenoidal
supercoiling proving the most effective in compacting the DNA. Solenoidal supercoiling is achieved with histones to form
a 10 nm fiber.
This fiber is further coiled into a 30 nm fiber, and further coiled upon itself numerous times more.
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18. Con’t
DNApackagingisgreatlyincreasedduringnucleardivisioneventssuchasmitosisormeiosis,where
DNAmustbecompactedandsegregatedtodaughtercells.
SupercoilingisalsorequiredforDNAandRNAsynthesis.BecauseDNAmustbeunwoundforDNA
andRNApolymeraseaction,supercoilswillresult
Quaternarystructure(4
0
):InteractionsofDNAandproteinssmallmoleculesarepresenttostabilize
thestructure.
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19. Forms of DNA
Threemajorforms:
B-DNA
A-DNA
Z-DNA
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20. B-DNA
ItisbiologicallyTHEMOSTCOMMON
Itisa-helixmeaningthatithasaRighthanded,orclockwise,spiral
IdealB-DNAhas10basepairperturn
Basepairare0.34nmapart.
Socompleterotationofmoleculeis3.4nm.
Axispassesthroughmiddleofeachbasepairs.
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21. B-DNA
MinorGrooveisNarrow,Shallow.
MajorGrooveisWide,Deep.
Thisstructureexistswhenplentyofwatersurroundsmoleculeandthereisnounusualbase
sequenceinDNA-Conditionthatarelikelytobepresentinthecells.
B-DNAstructureismoststableconfigurationforarandomsequenceofnucleotidesunder
physiologicalcondition.
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22. A-DNA
Right-handedhelix
WiderandflatterthanB-DNA
11bpperturn
Itsbasesaretiltedawayfrommainaxisofmolecule
NarrowDeepmajorGrooveandBroad,ShallowminorGroove.
Observedwhenlesswaterispresent.i.e.Dehydratingcondition.
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23. Z-DNA
Aleft-handedhelix
SeeninConditionofHighsaltconcentration.
Inthisformsugar-phosphatebackboneszigzagbackand
forth,givingrisetothenameZ-DNA(forzigzag).
12basepairsperturn.
AdeepMinorGroove.
NoDiscernibleMajorGroove.
PartofsomeactivegenesformZ-DNA,suggestingthatZ-
DNAmayplayaroleinregulatinggenetranscription.
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24. Summery on forms of DNA
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25. DNA Replication
CellsneedtomakeacopyofDNAbeforedividingsoeachdaughtercellhasacompletecopyofgenetic
information
DNAReplicationisthenormalprocessofdoublingtheDNAcontentofcellspriortonormalcell
division.
Becausethegeneticcomplementoftheresultantdaughtercellsmustbethesameastheparentalcell.
DNAreplicationmustpossessatveryhighdegreeoffidelity.
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26. Components of Replication
1.dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside5’-triphosphates) (sugar-base
+ 3 phosphates)
2.Ds DNA template
3.Primer-short RNA fragment with a free 3´-OH end
4.Enzyme: DNA-dependent DNA polymerase (DDDP), other enzymes, protein factor
5.Mg
2+
(optimizes DNA polymerase activity)
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27. Enzymes Involved in Replication
DNAhelicasesunwindthedoublehelix,thetemplatestrandsarestabilizedbyother
proteins
Single-strandedDNAbindingproteinsmakethetemplateavailable
RNAprimasecatalyzesthesynthesisofshortRNAprimers,towhichnucleotidesare
added.
DNApolymeraseIIIextendsthestrandinthe5’-to-3’direction
DNApolymeraseIdegradestheRNAprimerandreplacesitwithDNA
DNAligasejoinstheDNAfragmentsintoacontinuousdaughterstrand
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28. Unwind DNA
helicaseenzyme
unwinds part of DNA helix
stabilized by single-stranded binding proteins
prevents dnamolecule from closing!
DNA gyrase
Enzyme that prevents tangling upstream from the
replication fork
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29. RNA Primase
AddssmallsectionofRNA(RNAprimer)tothe3’endoftemplateDNA
Whymustthisbedone?
PrimasesynthesizesshortstretchesofRNAnucleotides,providinga3’-OHgrouptowhichDNA
polymerasecanaddDNAnucleotides
DNApolymerase3(enzymethatbuildsnewDNAstrand)canonlyaddnucleotidestoexistingstrandsof
DNA
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30. DNA Polymerase III
Build daughter DNA strand by adding new complementary bases
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31. DNA replication
DNAReplicationistheprocessbywhichtheDNAoftheancestralcellisduplicated,
priortocelldivision.
Uponcelldivision,eachofthedescendantswillgetonecompletecopyoftheDNAthatis
identicaltoitspredecessor
SynthesisofbothnewstrandsofDNAoccursatthereplicationforkthatmovesalong
theparentalmolecule
ThereplicationforkconsistsofthezoneofDNAwherethestrandsareseparated,plusan
assemblageofproteinsthatareresponsibleforsynthesis
Sometimesreferredtoasthereplisome
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32. DNA replication
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•ThereplicationforkisthesiteofDNAreplicationand,bydefinition,includes
boththeDNAandassociatedproteins

33. DNAreplicationinvolvesseveralprocesses:
First,theDNAmustbeunwound,separatingthetwostrands
Thesinglestrandsthenactastemplatesforsynthesisofthenewstrands,whichare
complimentaryinsequence
BasesareaddedoneatatimeuntiltwonewDNAstrandsthatexactlyduplicatethe
originalDNAareproduced
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DNA replication

34. Con’t
Theprocessiscalledsemi-conservative
replicationbecauseonestrandofeachdaughter
DNAcomesfromtheparentDNAandonestrand
isnew
Theenergyforthesynthesiscomesfrom
hydrolysisofphosphategroupsasthe
phosphodiesterbondsformbetweenthebases
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35. Direction of Replication
Theenzymehelicaseunwindsseveralsectionsof
parentDNA
AteachopenDNAsection,calledareplicationfork,
DNApolymerasecatalyzestheformationof5’-3’ester
bondsoftheleadingstrand
Thelaggingstrand,whichgrowsinthe3’-5’
direction,issynthesizedinshortsectionscalled
Okazakifragments
TheOkazakifragmentsarejoinedbyDNAligaseto
giveasingle3’-5’DNAstrand
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36. Mechanism of DNA Replication
DNAreplicationiscatalyzedbyDNApolymeraseIIIwhich
needsanRNAprimer
DNApolymeraseIIIcannotinitiatethesynthesisofa
polynucleotide,theycanonlyaddnucleotidestothe3end
TheinitialnucleotidestrandisanRNAprimer
RNAprimasesynthesizesprimeronDNAstrand
DNApolymeraseaddsnucleotidestothe3’endofthegrowing
strand
By: Asmamaw Menelih
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DNA polymerase I
degrades the RNA
primer and replaces it
with DNA
DNA polymerase III adds
nucleotides to primer

37. DNA Replication
By: Asmamaw Menelih
37
DNA polymerase I degrades the RNA primer and replaces it with DNA
DNA polymerase III adds nucleotides to primer

38. Mechanism of DNA Replication
Nucleotidesareaddedbycomplementarybasepairingwiththetemplatestrand
Duringreplication,newnucleotidesareaddedtothefree3’hydroxylonthegrowing
strand
Thenucleotides(deoxyribonucleosidetriphosphates)arehydrolyzedasadded,releasing
energyforDNAsynthesis.
Therateofelongationisabout500nucleotidespersecondinbacteriaand50persecond
inhumancells.
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39. 39
Mechanism of DNA Replication

40. The process of DNA replication
TheprocessofDNAreplicationfollowsthethreemainsteps:
1.Initiation,
2.Elongation,
3.Termination
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41. Initiation
Involvesrecognitionoftheoriginbyacomplexofproteins.
BeforeDNAsynthesisbegins,theparentalstrandsmustbeseparatedand(transiently)stabilizedin
thesingle-strandedstate.
ThensynthesisofdaughterstrandscanbeinitiatedatthereplicationforkbyRNAprimer.
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42. Elongation
Isundertakenbyanothercomplexofproteins.
Involves the addition of new nucleotides (dNTPs) based on
complementarity of the template strand
Forms phosphoesterbonds,
Correct the mismatch bases, extending the DNA strand
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DNA polymerase III

43. Termination
Attheendofthereplicon,joiningand/orterminationreactionsarenecessary.
Followingtermination,theduplicatechromosomesmustbeseparatedfromoneanother,whichrequires
manipulationofhigher-orderDNAstructure.
TheterminalstructureofeukaryoticDNAofchromosomesiscalledtelomere.
TelomereiscomposedofterminalDNASequenceandprotein
ThesequenceoftypicaltelomeresisrichinTandG
Thetelomerestructureiscrucialtokeeptheterminiofchromosomesinthecellfrombecoming
entangledandstickingtoeachother.
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45. Replication in prokaryotic Vs. eukaryotic
No.DANreplicationinprokaryotic DNAreplicationineukaryotic
1Itoccursinsidethecytoplasm Itoccursinsidethenucleus
2HaveOnlyoneoriginofreplicationperDNAmoleculeManyoriginofreplication(over1000)ineachchromosome
3Originofreplicationisformedofabout100-200ormore
nucleotides
Eachoriginofreplicationisformedofabout150nucleotides
4ReplicationofDNAoccursatonepointineachDNA
molecule
Occursatseveralpointssimultaneouslyineachchromosome
5Prokaryoticchromosomehasonereplicon
EukaryoticDNAmoleculeshavelargenumberofreplicons(50000and
above),butreplicationdoesnotoccursimultaneouslyonallreplicons
6OnereplicationbubbleisformedduringDNAreplicationNumerousreplicationbubblesareformedinonereplicatingDNAmolecule
7InitiationofDNAreplicationiscarriedoutbyprotein
DnaAandDnaB
Initiationiscarriedoutbymulti-sub-unitprotein,originrecognition
complex
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46. Cont..
8 DNAgyraseisneeded DNAgryaseisneeded
9 Okazakifragmentarelarge,1000-2000nucleotides
long
Okazakifragmentareshort,100-200nucleotideslong
10 Replicationisveryrapid,some2000bpsecondare
added
Replicationisslow,some100nucleotidespersecondareadded
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47. DNA mutation and repair
What is a mutation?
Changes in the nucleotide sequenceof DNA
May occur in somatic cells(aren’t passed to offspring)
May occur in gametes(eggs & sperm) and be passed to offspring
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48. Are Mutations Helpful or Harmful?
Mutations happen regularly
Almost all mutations are neutral
Chemicals & UVradiation cause mutations
Many mutations are repairedby enzymes
Some type of skin cancers and leukemiaresult from somaticmutations
Some mutations may improvean organism’s survival(beneficial)
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49. What is a mutation?
Substitution, deletion, or insertion of a base pair.
Chromosomal deletion, insertion, or rearrangement.
Somatic mutationsoccur in somatic cells and only affect the individual in which the mutation arises.
Germ-line mutationsalter gametes and passed to the next generation.
Mutations are quantified in two ways:
1.Mutation rate= probability of a particular type of mutation per unit time (or generation).
2.Mutation frequency= number of times a particular mutation occurs in a population of cells or
individuals.
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50. Type of Mutations
Transition:One purinereplaced by a different purine;orone pyrimidinereplaced
by a diferentpyrimidine
A G T C
Transversion:A purinereplaced by a pyrimidineor vice versa
I. Point mutation:
A. Base substitution
A T
Change in DNA
C G
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51. Type of Mutations …
B. Change in protein
1. Silent mutation:altered codoncodes for the same
a.a.
2. Neutral mutation:altered codoncodes for
functional similar a.a.
3. Missensemutation: altered codoncodes for
different dissimilar a.a.
4. Nonsense mutation:altered codon becomes a stop
codon
GAG (Glu) --->GAA (Glu)
GAG--->GAC (Asp) or (DE)
GAG ---> AAG (Lys)
GAG ---> UAG (stop)
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52. Type of Mutations …
Frameshift mutation: addition or deletion of one base-pair result in a shift of reading frame and
alter amino acid sequence
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53. Types of chromosomal mutations
Inversion
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54. Replication Fidelity
Replicationbasedontheprincipleofbasepairingiscrucialtothehighaccuracyof
geneticinformationtransfer.
Enzymesusetwomechanismstoensurethereplicationfidelity
Proofreadingandrealtimecorrection
Baseselection
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55. DNA repair mechanisms
Enzyme-based repair mechanisms prevent and repair mutations and damage to DNA in prokaryotes and
eukaryotes.
Types of mechanisms
DNA polymerase proofreading-3’-5’exonucleaseactivity corrects errors during the process of
replication.
Photoreactivation(also called light repair) -photolyaseenzyme is activated by UV light (320-370 nm)
and splits abnormal base dimersapart.
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56. Con’t
DemethylatingDNA repair enzymes-repair DNAs damaged by alkylation.
Nucleotide excision repair (NER)-Damaged regions of DNA unwind and are removed by specialized
proteins; new DNA is synthesized by DNA polymerase.
Methyl-directed mismatch repair-removes mismatched base regions not corrected by DNA polymerase
proofreading.
Sites targeted for repair are indicated in E. coliby the addition of a methyl (CH
3) group at a GATC
sequence.
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