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Structure and Structure and Replication Of Replication Of

DNA, DNA DNA, DNA damage & damage &

repairrepairDr. Madhumita BhattacharjeeAssiatant ProfessorBotany deptt.P.G.G.C.G. -11,Chandigarh

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History of DNAHistory of DNA Early scientists thought Early scientists thought proteinprotein

was the cell’s hereditary was the cell’s hereditary material because it was material because it was more more complexcomplex than DNA than DNA

Proteins were composed of Proteins were composed of 20 20 different amino acidsdifferent amino acids in long in long polypeptide chainspolypeptide chains

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TransformationTransformation Fred GriffithFred Griffith worked with worked with

virulent Svirulent S and and nonvirulent Rnonvirulent R strain strain PneumoccocusPneumoccocus bacteria bacteria

He found that He found that R strain could R strain could become virulentbecome virulent when it took in when it took in DNA from heat-killed S strainDNA from heat-killed S strain

Study Study suggested that DNA was suggested that DNA was probably the genetic materialprobably the genetic material

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Griffith ExperimentGriffith Experiment

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History of DNAHistory of DNA Chromosomes are Chromosomes are

made of both DNA made of both DNA and proteinand protein

Experiments on Experiments on bacteriophage bacteriophage viruses by Hershey viruses by Hershey & Chase proved that & Chase proved that DNA was the cell’s DNA was the cell’s genetic materialgenetic material

Radioactive 32P was injected into bacteria!

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Discovery of DNA Discovery of DNA StructureStructure

Erwin Chargaff showed the Erwin Chargaff showed the amountsamounts of the four bases on of the four bases on DNA ( A,T,C,G)DNA ( A,T,C,G)

In a body or somatic cell:In a body or somatic cell: A = 30.3%A = 30.3% T = 30.3%T = 30.3% G = 19.5%G = 19.5% C = 19.9%C = 19.9%

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Chargaff’s RuleChargaff’s Rule AdenineAdenine must pair with must pair with

ThymineThymine GuanineGuanine must pair with must pair with

CytosineCytosine The bases form weak The bases form weak

hydrogen bondshydrogen bonds

G CT A

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DNA StructureDNA Structure Rosalind FranklinRosalind Franklin took took

diffraction diffraction x-rayx-ray photographs of DNA photographs of DNA crystalscrystals

In the 1950’s, In the 1950’s, Watson Watson & Crick& Crick built the built the first first modelmodel of DNA using of DNA using Franklin’s x-raysFranklin’s x-rays

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DNADNA Two strands coiled called Two strands coiled called

a a double helixdouble helix SidesSides made of a pentose made of a pentose

sugar sugar Deoxyribose Deoxyribose bonded to phosphate bonded to phosphate (PO(PO44) groups by ) groups by phosphodiester bondsphosphodiester bonds

Center made of nitrogen Center made of nitrogen bases bonded together bases bonded together by by weak hydrogen bondsweak hydrogen bonds

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DNA Double HelixDNA Double Helix

NitrogenousNitrogenousBase (A,T,G or C)Base (A,T,G or C)

““Rungs of ladder”Rungs of ladder”

““Legs of ladder”Legs of ladder”

Phosphate &Phosphate &Sugar BackboneSugar Backbone

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HelixHelix

Most DNA has a Most DNA has a right-right-hand twist with 10 base hand twist with 10 base pairspairs in a complete turn in a complete turn

Left twisted DNA is called Left twisted DNA is called Z-DNA Z-DNA or or southpawsouthpaw DNA DNA

Hot spotsHot spots occur where occur where right and left twisted right and left twisted DNA meet producingDNA meet producing mutationsmutations

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DNADNA

Stands for Stands for Deoxyribonucleic acidDeoxyribonucleic acid

Made up of subunits Made up of subunits called called nucleotidesnucleotides

NucleotideNucleotide made of: made of:1.1. Phosphate groupPhosphate group2.2. 5-carbon sugar5-carbon sugar3.3. Nitrogenous baseNitrogenous base

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DNA NucleotideDNA Nucleotide

O=P-O O

PhosphatePhosphate GroupGroup

NNitrogenous baseNitrogenous base (A, G, C, or T)(A, G, C, or T)

CH2

O

C1C4

C3 C2

5

SugarSugar(deoxyribose)(deoxyribose)

O

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Pentose SugarPentose Sugar Carbons are numbered Carbons are numbered

clockwise 1’ to 5’clockwise 1’ to 5’CH2

O

C1C4

C3 C2

5

SugarSugar(deoxyribose)(deoxyribose)

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DNADNA

P

P

P

O

O

O

1

23

4

5

5

3

3

5

P

P

PO

O

O

1

2 3

4

5

5

3

5

3

G C

T A

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Antiparallel Antiparallel StrandsStrands

One strand of One strand of DNA goes from DNA goes from 5’ to 3’ (sugars)5’ to 3’ (sugars)

The other The other strand is strand is opposite in opposite in directiondirection going going 3’ to 5’ (sugars)3’ to 5’ (sugars)

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Nitrogenous Nitrogenous BasesBases

Double ring Double ring PURINESPURINESAdenine (A)Adenine (A)Guanine (G)Guanine (G)

Single ring Single ring PYRIMIDINESPYRIMIDINES

Thymine (T)Thymine (T)Cytosine (C)Cytosine (C) T or C

A or G

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Base-PairingsBase-Pairings Purines only pair with Purines only pair with

PyrimidinesPyrimidines ThreeThree hydrogen bonds hydrogen bonds

required to bond required to bond Guanine & CytosineGuanine & Cytosine

CG

3 H-bonds

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T A

•Two Two hydrogen bonds hydrogen bonds are required to bond are required to bond Adenine & ThymineAdenine & Thymine

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DNA DNA ReplicationReplication

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Replication FactsReplication Facts

DNA has to be copied DNA has to be copied before a cell dividesbefore a cell divides

DNA is copied during DNA is copied during the the S S or synthesis phase or synthesis phase of of interphaseinterphase

New cells will need New cells will need identical identical DNA strandsDNA strands

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Semiconservative Model Semiconservative Model of Replicationof Replication

Idea presented by Idea presented by Watson & CrickWatson & Crick The two strands of the parental The two strands of the parental

molecule separate, and each acts molecule separate, and each acts as a as a templatetemplate for a for a new new complementary strandcomplementary strand

New DNA consists of 1 New DNA consists of 1 PARENTAL (original) and 1 PARENTAL (original) and 1 NEW strand of DNANEW strand of DNA

Parental DNA

DNA Template

New DNA

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Synthesis Phase (S Synthesis Phase (S phase)phase)

S phase during S phase during interphase interphase of of the cell cyclethe cell cycle

Nucleus of eukaryotesNucleus of eukaryotes

Mitosis-prophase-metaphase-anaphase-telophase

G1 G2

Sphase

interphase

DNA replication takesDNA replication takesplace in the S phase.place in the S phase.

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DNA ReplicationDNA Replication

Begins atBegins at Origins of ReplicationOrigins of Replication Two strands open forming Two strands open forming

Replication Forks (Y-shaped Replication Forks (Y-shaped region)region)

New strands grow at the forksNew strands grow at the forks

ReplicationReplicationForkFork

Parental DNA MoleculeParental DNA Molecule

3’

5’

3’

5’

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DNA ReplicationDNA Replication As the 2 DNA strands open at As the 2 DNA strands open at

the origin, the origin, Replication Replication Bubbles formBubbles form

ProkaryotesProkaryotes (bacteria) have (bacteria) have a a singlesingle bubble bubble

EukaryoticEukaryotic chromosomes chromosomes have have MANYMANY bubbles bubbles

Bubbles Bubbles

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DNA ReplicationDNA Replication Enzyme Enzyme HelicaseHelicase

unwinds and separates unwinds and separates the 2 DNA strands by the 2 DNA strands by breaking the breaking the weak weak hydrogen bondshydrogen bonds

Single-Strand BindingSingle-Strand Binding ProteinsProteins attach and attach and keep the 2 DNA strands keep the 2 DNA strands separated and separated and untwisteduntwisted

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DNA ReplicationDNA Replication Enzyme Enzyme Topoisomerase Topoisomerase

attaches to the 2 forks of attaches to the 2 forks of the bubble to the bubble to relieve stressrelieve stress on the DNA molecule as it on the DNA molecule as it separatesseparatesEnzyme

DNA

Enzyme

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DNA ReplicationDNA Replication Before new DNA strands can Before new DNA strands can

form, there must be form, there must be RNA RNA primersprimers present to start the present to start the addition of new nucleotidesaddition of new nucleotides

PrimasePrimase is the enzyme that is the enzyme that synthesizes the RNA Primersynthesizes the RNA Primer

DNA polymeraseDNA polymerase can then can then add the new nucleotidesadd the new nucleotides

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DNA ReplicationDNA Replication DNA polymeraseDNA polymerase can only add can only add

nucleotides to the nucleotides to the 3’ end3’ end of of the DNA the DNA

This causes the This causes the NEW strand to NEW strand to be built in a 5’ to 3’ directionbe built in a 5’ to 3’ direction

RNARNAPrimerPrimerDNA PolymeraseDNA Polymerase

NucleotideNucleotide

5’

5’ 3’

Direction of ReplicationDirection of Replication

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Synthesis of the New Synthesis of the New DNA StrandsDNA Strands

The The Leading StrandLeading Strand is is synthesized as a single synthesized as a single strand from the strand from the point of point of origin toward the opening origin toward the opening replication forkreplication fork

RNARNAPrimerPrimerDNA PolymeraseDNA PolymeraseNucleotidesNucleotides

3’5’

5’

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Synthesis of the New DNA Synthesis of the New DNA StrandsStrands

The The Lagging Strand is synthesized Lagging Strand is synthesized discontinuously againstdiscontinuously against overall overall direction of replicationdirection of replication

This strand is made in This strand is made in MANY short MANY short segmentssegments It is replicated It is replicated from the from the replication fork toward the originreplication fork toward the origin

RNA PrimerRNA Primer

Leading StrandLeading Strand

DNA PolymeraseDNA Polymerase

5’

5’

3’

3’

Lagging StrandLagging Strand

5’

5’

3’

3’

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Lagging Strand Lagging Strand SegmentsSegments

Okazaki FragmentsOkazaki Fragments - - series series of short segments on the of short segments on the lagging strandlagging strand

Must be joined together by Must be joined together by an an enzymeenzyme

Lagging Strand

RNARNAPrimerPrimer

DNADNAPolymerasePolymerase

3’

3’

5’

5’

Okazaki FragmentOkazaki Fragment

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Joining of Okazaki Joining of Okazaki FragmentsFragments

The enzyme The enzyme LigaseLigase joins the joins the Okazaki fragments together Okazaki fragments together to make one strandto make one strand

Lagging Strand

Okazaki Fragment 2Okazaki Fragment 2

DNA ligaseDNA ligase

Okazaki Fragment 1Okazaki Fragment 1

5’

5’

3’

3’

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Replication of Replication of StrandsStrands

Replication Fork

Point of Origin

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Proofreading New Proofreading New DNADNA

DNA polymerase initially DNA polymerase initially makes about makes about 1 in 10,0001 in 10,000 base base pairing errorspairing errors

Enzymes Enzymes proofread and proofread and correct these mistakescorrect these mistakes

The new error rate for DNA The new error rate for DNA that has been proofread is that has been proofread is 1 1 in 1 billion base pairing errorsin 1 billion base pairing errors

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DNA Damage & DNA Damage & RepairRepair

Chemicals &Chemicals & ultraviolet radiationultraviolet radiation damage the DNA in our body cellsdamage the DNA in our body cells

Cells must Cells must continuouslycontinuously repair repair DAMAGED DNADAMAGED DNA

Excision repair occurs when any of Excision repair occurs when any of over 50 repairover 50 repair enzymes remove enzymes remove damaged parts of DNAdamaged parts of DNA

DNA polymerase and DNA ligaseDNA polymerase and DNA ligase replace and bond the new replace and bond the new nucleotides togethernucleotides together

UV radiation causes UV radiation causes thymine dimers, thymine dimers, which block which block replication.replication.

Light-repair Light-repair separates thymine separates thymine dimersdimers

Sometimes the Sometimes the “repair job” “repair job” introduces the introduces the wrong nucleotide, wrong nucleotide, leading to a point leading to a point mutation.mutation.

Ionizing Radiation: UVIonizing Radiation: UV

Figure 8.20

Mismatch and SOS/”Light” Repair: Error ProneMismatch and SOS/”Light” Repair: Error Prone

RecA

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