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Chapter 16.

DNAThe Genetic Material

Replication

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Scientific History The march to understanding that DNA

is the genetic material T.H. Morgan (1908) Frederick Griffith (1928) Avery, McCarty & MacLeod (1944) Hershey & Chase (1952) Watson & Crick (1953) Meselson & Stahl (1958)

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Genes are on chromosomes T.H. Morgan

working with Drosophila (fruit flies) genes are on chromosomes but is it the protein or the DNA of the

chromosomes that are the genes? through 1940 proteins were thought to be

genetic material… Why?

1908|1933

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The “Transforming Factor” Frederick Griffith

Streptococcus pneumonia bacteria was working to find cure for pneumonia

harmless live bacteria mixed with heat-killed infectious bacteria causes diseasein mice

substance passed from dead bacteria tolive bacteria = “Transforming Factor”

1928

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The “Transforming Factor”

Transformation: change in genotype & phenotypedue to assimilation of external DNA by a cell

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DNA is the “Transforming Factor” Avery, McCarty & MacLeod

purified both DNA & proteins fromStreptococcus pneumonia bacteria which will transform non-pathogenic bacteria?

injected protein into bacteria no effect

injected DNA into bacteria transformed harmless bacteria into virulent

bacteria

What’s the conclusion?

1944

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Avery, McCarty & MacLeod

Oswald Avery

Maclyn McCarty

Colin MacLeod

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Confirmation of DNA Hershey & Chase

classic “blender” experiment worked with bacteriophage

viruses that infect bacteria grew phage viruses in 2 media,

radioactively labeled with either: 35S in their proteins 32P in their DNA

infected bacteria withlabeled phages

1952|1969

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Hershey & Chase

Alfred HersheyMartha Chase

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Blender experiment Radioactive phage & bacteria in blender

35S phage radioactive proteins stayed in supernatant therefore protein did NOT enter bacteria

32P phage radioactive DNA stayed in pellet therefore DNA did enter bacteria

DNA is confirmed as “transformingfactor”

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Chargaff DNA composition: “Chargaff’s rules”

varies from species to species amounts of 4 bases not equal bases present in characteristic ratio

humans: A=30.9% T=29.4% G=19.9% C=19.8%

1947

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Structure of DNA Watson & Crick

developed double helix model of DNA other scientists working on question:

Linus PaulingMaurice WilkinsRosalind Franklin

1953|1962

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Rosalind Franklin (1920-1958)

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Watson and Crick 1953 article in Nature

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Double helix structure of DNA

the structure of DNA suggested a mechanism forhow DNA is copied by the cell

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Anti-parallel strands Phosphate to sugar bond

involves carbons in 3’ & 5’positions DNA molecule has

“direction” complementary strand

runs in opposite direction

“It has not escaped our notice thatthe specific pairing we havepostulated immediately suggests apossible copying mechanism for thegenetic material.”

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Bonding in DNAhydrogen

bonds

….strong or weak bond?How do the bonds fit the mechanism for copying DNA?

3’

5’ 3’

5’

phosphodiesterbonds

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Base pairing in DNA Purines

adenine guanine

Pyrimidines thymine cytosine

Pairing A : T C : G

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Copying DNA Replication of DNA

base pairing allowseach strand to serveas a pattern for anew strand

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Models of DNA Replication Alternative models

needed to be verified through experimentation

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Semi-conservative replication Meselson & Stahl

labeled nucleotides of “parent” DNA strandswith heavy nitrogen = 15N

labeled new nucleotides with lighter isotope =14N

replicated strands were found to be half 15N &half 14N

“The Most Beautiful Experiment in Biology”

1958

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DNA Replication Large team of enzymes coordinates replication

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Helicase Opens DNA helix enabling replication

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DNA Polymerase III Adds nucleotides only to 3’ end

nucleoside-P-P-P links to sugar-P backbone losing 2-P provides energy for bonding

Does this sound familiar??

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A common thread in biology…

5’

3’

3’

5’

You don’t justget energy fromATP → ADP

Why 3Ps?

TTP → TMPGTP→ GMPCTP → CMP

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Leading & Lagging strands

Leading strand continuous synthesis

Lagging strand Okazaki fragments joined by ligase

“spot welder”

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Okazaki fragments

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Priming DNA synthesisDNA polymerasecan only extend anexisting DNAmolecule. Cannotstart a new one. short RNA primer

is built first onparent DNA strandby primase

RNA primer laterremoved by DNApolymerase I

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Replication fork

3’

5’3’

5’

5’

3’

leading strand

Okazaki fragments

lagging strand

3’ 5’

DNA polymerase

ligase

helicase

direction of replication

primase

DNA polymerase

SSB

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Replication bubbleAdds 1000 bases/second!

Which direction does DNA build?List the enzymes & their role

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Replication enzymes

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DNA polymerases DNA polymerase I

20 bases/second editing, repair & primer removal

DNA polymerase III 1000 bases/second main DNA building enzyme

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Editing & proofreading DNA At 1000

bases/second,lots of typos!

DNA polymerase Iexcisesmismatched bases reduces error rate

from 1 in 10,000 to1 in 100 millionbases

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Fast & accurate! It takes E. coli <1 hour to copy

5 million base pairs in its singlechromosome & divide to form 2identical daughter cells

Human cell copies its 6 billion bases &divide into daughter cells in only fewhours remarkably accurate only ~1 error per 100 million bases ~30 errors per cell cycle

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Problems in the end Ends of

chromosomesare erodedwith eachreplication

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Telomeres expendable,

non-codingsequences atends of DNA short sequence

of basesrepeated 1000stimes

TTAGGG inhumans

Telomeraseenzyme in certaincells

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proteinRNA

The “Central Dogma”

DNAtranscription translation

replication

flow of genetic information within a cell