DNADNA andand ReplicationReplication

1 HistoryHistory ofof DNADNA

2 History of DNA

• Early scientists thought protein was the cell’s hereditary material because it was more complex than DNA • Proteins were composed of 20 different amino acids in long polypeptide chains

3 TransformationTransformation • Fred Griffith worked with virulent S and nonvirulent R strain Pneumoccocus bacteria • He found that R strain could become virulent when it took in DNA from heat-killed S strain • Study suggested that DNA was probably the genetic material

4 Griffith Experiment

5 History of DNA • Chromosomes are made of both DNA and protein • Experiments on bacteriophage viruses by Hershey & Chase proved that DNA was the cell’s genetic material

Radioactive 32P was injected into bacteria! 6 DiscoveryDiscovery ofof DNADNA StructureStructure • Erwin Chargraff showed the amounts of the four bases on DNA ( A,T,C,G) • In a body or somatic cell: A = 30.3% T = 30.3% G = 19.5% C = 19.9%

7 ChargaffChargaff’’ss RuleRule •• AdenineAdenine must pair with ThymineThymine •• GuanineGuanine must pair with CytosineCytosine • The bases form weak hydrogen bonds

T A G C

8 DNADNA StructureStructure • Rosalind Franklin took diffraction x-ray photographs of DNA crystals • In the 1950’s, Watson & Crick built the first model of DNA using Franklin’s x-rays

9 RosalindRosalind FranklinFranklin

10 DNADNA StructureStructure

11 DNADNA • Two strands coiled called a double helix • Sides made of a pentose sugar Deoxyribose bonded to phosphate (PO4) groups by phosphodiester bonds • Center made of nitrogen bases bonded together by weak hydrogen bonds 12 DNADNA DoubleDouble HelixHelix

“Rungs of ladder”

Nitrogenous Base (A,T,G or C)

“Legs of ladder”

Phosphate & Sugar Backbone

13 HelixHelix

•• MostMost DNADNA hashas aa rightright--handhand twisttwist withwith 1010 basebase pairspairs inin aa completecomplete turnturn •• LeftLeft twistedtwisted DNADNA isis calledcalled ZZ --DNADNA oror southpawsouthpaw DNADNA •• HotHot spotsspots occuroccur wherewhere rightright andand leftleft twistedtwisted DNADNA meetmeet producingproducing mutationsmutations

14 DNADNA • Stands for Deoxyribonucleic acid • Made up of subunits called nucleotidesnucleotides •• NucleotideNucleotide mademade of:of: 1. PhosphatePhosphate groupgroup 2. 55 --carboncarbon sugarsugar 3. NitrogenousNitrogenous basebase 15 DNADNA NucleotideNucleotide Phosphate Group

O 5 O=P-O CH2 O O N Nitrogenous base 1 Nitrogenous base C4 C (A, G, C, or T) Sugar (deoxyribose) (deoxyribose) 16 C3 C2 PentosePentose SugarSugar • Sugars are numbered clockwise

1’ to 5’ 5 CH2

O

C4 C1 Sugar (deoxyribose) C3 C2

17 DNADNA 5 O 3

3 O P 5 P 5 O C 1 G 2 3 4 4 2 1 3 5 P O 3 P 5 T A O

O 5 P 3 18P AntiparallelAntiparallel StrandsStrands

• One strand of DNA goes from 5’ to 3’ (sugars) • The other strand is opposite in direction going 3’ to 5’ (sugars)

19 NitrogenousNitrogenous BasesBases •• DoubleDouble ringring PURINESPURINES AdenineAdenine (A)(A)

GuanineGuanine (G)(G) A or G

•• SingleSingle ringring PYRIMIDINESPYRIMIDINES ThymineThymine (T)(T) CytosineCytosine (C)(C) T or C

20 BaseBase--PairingsPairings • Purines only pair with Pyrimidines • Three hydrogen bonds required to bond Guanine & Cytosine 3 H-bonds

G C

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

T A

22 Question:Question:

• If there is 30% AdenineAdenine , how much CytosineCytosine is present?

23 Answer:Answer: • There would be 20% CytosineCytosine •• AdenineAdenine (30%)(30%) == ThymineThymine (30%)(30%) •• GuanineGuanine (20%)(20%) == CytosineCytosine (20%)(20%) •• Therefore,Therefore, 60%60% AA--TT andand 40%40% CC--GG

24 DNADNA ReplicationReplication

25 Replication of DNA

• DNA is the only molecule that can replicate itself. This is accomplished through a process known as replication.

26 • They attach themselves at their complementary bases.

• A series of enzymes called polymearases fuse the free nucleotides together in the complimentary chain of DNA.

• The free floating nucleotides in your cells are derived from the food you eat. Steak supplies you with muscle cells from a cow. Does not mean you will turn into a cow. Specialized enzymes in your digestive tract break down the cow DNA into cucleotides which you use to make human DNA 27 • Genetic mistakes do occur but are very infrequent

• Environmental factors such as hazardous chemicals or radiation are one source of genetic mistakes that do occur.

• Can cause uncomplimentary bases to join.

• Permanent damage is prevented by enzyme that act as proof readers. They run along the strands of DNA looking for mismatched pairs. It snips the error and replaces it with the correct nucleotide.

28 Replication • the weak hydrogen bonds that hold the complementary nitrogen bases together are broken. The two edges of the ladder seem to unzip.

• The parent strands are conserved (remain intact)

• This is called semiconservative replication

• semiconservative replication produces two “half old, half new” strands of DNA.

• Each parent strand acts as a template or mold to which free floating nucleotides in29 the cell can attach.

• LAB page 525 - photocopy required

30 Lab

31 ReplicationReplication FactsFacts

•• DNADNA hashas toto bebe copiedcopied beforebefore aa cellcell dividesdivides •• DNADNA isis copiedcopied duringduring thethe SS oror synthesissynthesis phasephase ofof interphaseinterphase •• NewNew cellscells willwill needneed identicalidentical DNADNA strandsstrands

32 SynthesisSynthesis PhasePhase (S(S phase)phase) • S phase during interphase of the cell cycle • Nucleus of eukaryotes S DNA replication takes phase place in the S phase. G1 interphase G2

Mitosis -prophase -metaphase -anaphase -telophase 33 DNADNA ReplicationReplication

•• BeginsBegins atat OriginsOrigins ofof ReplicationReplication •• TwoTwo strandsstrands openopen formingforming ReplicationReplication ForksForks (Y(Y--shapedshaped region)region) •• NewNew strandsstrands growgrow atat thethe forksforks 3’

Parental DNA Molecule 5’ Replication Fork 3’

34 5’ DNADNA ReplicationReplication

•• AsAs thethe 22 DNADNA strandsstrands openopen atat thethe origin,origin, ReplicationReplication BubblesBubbles formform • Eukaryotic chromosomes have MANY bubbles • Prokaryotes (bacteria) have a single bubble

Bubbles Bubbles

35 DNADNA ReplicationReplication •• EnzymeEnzyme HelicaseHelicase unwindsunwinds andand separatesseparates thethe 22 DNADNA strandsstrands byby breakingbreaking thethe weakweak hydrogenhydrogen bondsbonds •• SingleSingle--StrandStrand BindingBinding ProteinsProteins attach and keep the 2 DNA strands separated and untwisted

36 DNADNA ReplicationReplication •• EnzymeEnzyme TopoisomeraseTopoisomerase attaches to the 2 forks of the bubble to relieverelieve stressstress on the DNADNA moleculemolecule as it separates

Enzyme Enzyme

DNA

37 DNADNA ReplicationReplication •• BeforeBefore new DNA strands can form, there must be RNARNA primersprimers present to start the addition of new nucleotides •• PrimasePrimase is the enzyme that synthesizes the RNA Primer • DNA polymerase can then add the new nucleotides

38 39 DNADNA ReplicationReplication •• DNADNA polymerasepolymerase cancan onlyonly addadd nucleotidesnucleotides toto thethe 33’’ endend ofof thethe DNADNA •• ThisThis causescauses thethe NEWNEW strandstrand toto bebe builtbuilt inin aa 55’’ toto 33’’ directiondirection 5’ 3’

5’ RNA DNA Polymerase Nucleotide Primer

Direction of Replication 40 RememberRemember HOWHOW thethe CarbonsCarbons AreAre Numbered!Numbered! Phosphate Group

O 5 CH2 O=P-O O O N Nitrogenous base 4 C1 C (A, G, C, or T) Sugar (deoxyribose) 3 2 C C 41 RememberRemember thethe StrandsStrands areare

5 AntiparallelAntiparallel O 3

3 O P 5 P 5 O C 1 G 2 3 4 4 2 1 3 5 P O 3 P 5 T A O

O 5 P 3 42P SynthesisSynthesis ofof thethe NewNew DNADNA StrandsStrands •• TheThe LeadingLeading StrandStrand is synthesized as a singlesingle strandstrand from the point of origin toward the opening replication fork

5’ 3’ 5’ RNA Nucleotides DNA Polymerase Primer

43 SynthesisSynthesis ofof thethe NewNew DNADNA StrandsStrands • The Lagging Strand is synthesized discontinuously against overall direction of replication • This strand is made in MANY short segments It is replicated from the replication fork toward the origin

5’ Leading Strand 3’

3’ 5’ DNA Polymerase RNA Primer 5’ 3’

3’ 445’ Lagging Strand LaggingLagging StrandStrand SegmentsSegments •• OkazakiOkazaki FragmentsFragments -- series of short segments on the lagginglagging strandstrand •• MustMust bebe joinedjoined togethertogether byby anan

enzymeenzyme DNA Okazaki Fragment Polymerase RNA Primer 5’ 3’

3’ 5’ Lagging Strand 45 JoiningJoining ofof OkazakiOkazaki FragmentsFragments

•• TheThe enzymeenzyme LigaseLigase joinsjoins thethe OkazakiOkazaki fragmentsfragments togethertogether toto makemake oneone strandstrand

DNA ligase Okazaki Fragment 1 Okazaki Fragment 2 5’ 3’

3’ 5’ Lagging Strand

46 ReplicationReplication ofof StrandsStrands

Replication Point of Origin Fork

47 ProofreadingProofreading NewNew DNADNA

•• DNADNA polymerasepolymerase initiallyinitially makesmakes aboutabout 11 inin 10,00010,000 basebase pairingpairing errorserrors •• EnzymesEnzymes proofreadproofread andand correctcorrect thesethese mistakesmistakes •• TheThe newnew errorerror raterate forfor DNADNA thatthat hashas beenbeen proofreadproofread isis 11 inin 11 billionbillion basebase pairingpairing errorserrors

48 SemiconservativeSemiconservative ModelModel ofof ReplicationReplication •• IdeaIdea presentedpresented byby WatsonWatson && CrickCrick •• TheThe two strands of the parental molecule separate, and each acts as a template for a new complementary strand • New DNA consists of 1 PARENTAL (original) and 1 NEW strand of DNA DNA Template Parental DNA New DNA

49 DNADNA DamageDamage && RepairRepair • Chemicals & ultraviolet radiation damage the DNA in our body cells • Cells must continuously repair DAMAGED DNA • Excision repair occurs when any of over 50 repair enzymes remove damaged parts of DNA • DNA polymerase and DNA ligase replace and bond the new nucleotides together

50 Question:Question:

• What would be the complementary DNA strand for the following DNA sequence?

DNADNA 55’’--CGTATGCGTATG--33’’

51 Answer:Answer:

DNADNA 55’’--GCGTATGGCGTATG--33’’ DNADNA 33’’--CGCATACCGCATAC--55’’

52 53