1 Structure and Structure and Replication Of Replication Of DNA, DNA damage DNA, DNA damage & repair & repair Dr. Madhumita Bhattacharjee Assiatant Professor Botany deptt. P.G.G.C.G. -11,Chandigarh
Jan 02, 2016
1
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
2
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
3
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
4
Griffith ExperimentGriffith Experiment
5
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!
6
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%
7
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
8
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
9
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
10
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
11
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
12
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
13
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
14
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)
15
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
16
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)
17
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
18
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
19
T A
•Two Two hydrogen bonds hydrogen bonds are required to bond are required to bond Adenine & ThymineAdenine & Thymine
20
DNA DNA ReplicationReplication
21
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
22
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
23
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.
24
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’
25
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
26
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
27
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
28
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
29
30
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
31
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’
32
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’
33
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
34
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’
35
Replication of Replication of StrandsStrands
Replication Fork
Point of Origin
36
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
37
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
ThanksThanks