CAMPBELL AND REECECHAPTER 16

The Molecular Basis of Inheritance

The Search for Genetic Material

once Morgan proved genes are in chromosomes big debate started:

Is the genetic material in chromosomes the DNA or the proteins?

@ first case for proteins seemed stronger very heterogenous great specificty

Evidence that DNA can Transform Bacteria

1928 Griffith studied Streptococcus pneumoniae

Transformation

term coined by Griffithchange in genotype & phenotype due to

the assimilation of external DNA by a cell

Avery spent the next 14 years identifying the “transforming agent”

Avery’s Experiment

Avery

& his colleagues announced DNA was the transforming agent

many were skeptical Was bacterial DNA anything like eukaryotic

DNA? Nothing much known about DNA Most scientists held to belief that proteins

had to be transforming agent

Bacteriophages

are viruses that infect bacteria“phages” for shortVirus made of a protein coat covering

genetic material to produce more viruses it must invade a cell & take over the cell’s metabolic machinery

Hershey & Chase Experiment

Additional Evidence that DNA is Genetic Material

Chargraff already knew DNA made up of:

Deoxyribose Phosphate group Nitrogenous Base (A, G, C, T)

analyzed DNA from # of species 1950: base composition of DNA varies

between species made DNA more credible

Chargraff’s Rule

no matter what the source of DNA tested:

What is the structure of DNA?

early 1950’s: scientists convinced DNA carried genetic info

focus now on DNA’s structure

knew arrangement of DNA’s covalent bonds

Watson & Crick

Cambridge, England2 young, unknown scientistssame lab: Franklin & Wilkins doing x-ray

crystallography on protein structure

X-Ray Crystallography of DNA

Rosalind Franklin had purified some DNA and showed results to Watkins who was familiar with pattern made by a helical structure

Watson & Crick

began building models that satisfied: known chemical properties of DNA

nitrogenous bases relatively hydrophobic phosphate groups carry (-) charge

Chagraff’s rules helical structure how could this structure pass on genetic

information?

DNA Structure

antiparallel: arrangement of sugar-phosphate backbones in a DNA double helix

means 1 strand runs 5’ 3’ going “up” * the other runs 5’ 3’ going “down”

DNA Structure

because of size differences in dbl ringed purines vs. single ringed pyrimidines Watson & Crick knew could not have a purine linked with itself or the other purine

also knew that adenine & thymine could form H bonds (2) with each other & cytosine & guanine could for 3 H bonds

Watson & Crick

their 1 page paper published in Nature in April 1953

Watson, Crick, and Wilkins received Nobel Prize in 1962 (Franklin died in 1958)

DNA Replication

Watson and Crick’s 2nd paper stated their hypothesis on how DNA replicates:

DNA model is pair of complimentary templates

prior to replication H bonds broken & chains separate & unwind

each chain then acts as template for formation onto itself of a new complimentary chain

allows for exact duplication

DNA Replication

Watson & Crick’s Semiconservative Model of DNA Replication

predicts when a dbl helix replicates, each of the 2 daughter molecules will have 1 old strand and 1 new strand

Conservative Model: 1 new daughter molecule with 2 new strands & the original molecule

Dispersive Model: all 4 strands of DNA after replication have mixture of old & new parts

3 Models of DNA Replication

DNA Replication

begins @ particular sites called:Origins of Replication

short stretches of a specific sequence of nucleotides

many bacterial loops of DNA have single origin

proteins that initiate DNA replication recognize the sequence / attach to the DNA / separate the 2 strands by breaking H bonds creating “bubbles”

Prokaryotic Replication of DNA

Eukaryotic DNA Replication

Replication Bubble

Replication Forks

@ each end of the replication bubbleY-shaped region where DNA is unwindingproteins that participate in the unwinding:1. helicases

unwind double helix

2. single-strand binding proteins bind to single strands prevents them from

rewinding

3. topoisomerases untwisting dbl helix puts strain on ahead of

replication fork, these proteins relieve strain by breaking, swiveling, & rejoining DNA strands

Replication Forks

Replication of DNA

initial nucleotide chain made during DNA synthesis is actually a strand of RNA

this RNA chain called a primer which is made by an enzyme called primase (last slide)

primase starts a complementary RNA chain from a single RNA nucleotide then adds 1 @ time

Primers

when primer 5 – 10 nucleotides long...new DNA strand will start from the 3’ end of the RNA primer

DNA Polymerase

enzyme that catalyzes the synthesis of new DNA by adding nucleotides to a pre-existing chain 2 major one in prokaryotes 11 different ones in eukaryotes

most require a primer & DNA template strand

rate: ~500 nucleotides/s in bacteria ~ 50 nucleotides/s in human cells

Source of Nucleotides

are in form of nucleoside triphosphates

dATP

Nucleoside Triphosphates

are chemically reactive (like ATP, except sugar is deoxyribose, not ribose)

as each nucleotide joins the growing end of a DNA strand 2 of the phosphate groups are lost as a molecule of inorganic phosphate in a couple exergonic reaction that drives the polymerization reaction

Polymerization Reaction

Antiparallel Elongation

each strand of DNA has directionality (1-way street)

& each strand oriented in opposite directions to each other

DNA polymerase III can add nucleotides only to the free 3’ end of a primer or growing DNA strand along 1 template DNA polymerase

synthesizes complementary strand continuously (5’ 3’ direction)

called the Leading Strand

Antiparallel Elongation

along opposite strand because of orientation, DNA polymerase III must work in direction away from the replication fork

called Lagging Strand synthesized in short segments called:

Okazaki Fragments ~ 1,000 – 2,000 nucleotides long in E. coli ~ 100 – 200 nucleotides long in eukaryotes

DNA Replication Complex

easy to think of DNA polymerase as a locomotive moving down template track but not really how it works:

1. various proteins that participate in DNA replication form a large complex

2. DNA replication complex doesn’t move, the DNA template moves thru the complex

DNA Replication Complex

Proofreading & Repairing DNA

~ 1/10 billion base pairs in completed DNA will be incorrect

but right after strands replicated errors ~ 100,000 times more common

DNA polymerases “proofread” each nucleotide against its template as soon as it is added

when error found, incorrect nucleotide removed, correct 1 inserted

Proofreading & Repairing

some errors evade DNA polymerase…other enzymes remove & replace incorrectly paired nucleotides

some errors arise after replication: damage to DNA relatively common: usually corrected by b/4 becoming permanent mutations

cells continuously monitor & repair damaged DNA

Repair Enzymes

~100 in E. coli~ 130 in humansmost organisms use same mechanism to

repair errors or damage involves cutting out damaged area using

DNA-cutting enzyme called nuclease gap then filled with correct nucleotides done

by a DNA polymerase & DNA ligase 1 of these systems called nucleotide excision

repair

Nucleotide Excision Repair

Telomeres

repetitive sequences @ ends of eukaryotic chromosomes shorter as we age (with each round of DNA

replication) so preserves the ends of linear DNA &

postpones erosion of genes telomerase catalyzes the lengthening of

telomeres in germ cells

Telomeres

Prokaryotic DNA

usually loop of DNAsome associated proteinsloop of DNA + proteins = nucleoid

Eukaryotic Chromatin

includes:1. DNA2. histones3. other proteins

Nucleosomes

histones bind to each other & to the DNA to form nucleosomes: the most basic unit of DNA packing

histone tails extend outward from each bead-like nucleosome cone

additional coiling & folding highly condensed chromosome as seen in mitosis

Euchromatin: term for the less coiled chromatin seen in interphase cells easily accessible for transcription

Heterochromatin: portions of chromatin that remains highly condensed even in interphase mostly inaccessible to transcription