Chapter 16 The Molecular Basis

of Inheritance

Question?

• Traits are inherited on

chromosomes, but what in the

chromosomes is the genetic

material?

• Two possibilities:• Protein

• DNA

Qualifications

• Protein:• very complex.

• high specificity of function.

• DNA:• simple.

• not much known about it (early

1900’s).

For testing:

• Name(s) of experimenters

• Outline of the experiment

• Result of the experiment and

the importance of the result

Griffith - 1928

• Pneumonia in mice.

• Two strains:• S - pathogenic

• R - harmless

Griffith’s Experiment

Result

• Something turned the R cells

into S cells.

• Transformation - the

assimilation of external genetic

material by a cell.

Problem

• Griffith used heat.

• Heat denatures proteins.

• So could proteins be the genetic material?

• DNA - heat stable.

• Griffith’s results contrary to accepted views.

Avery, McCarty and MacLeod

- 1944

• Repeated Griffith’s experiments,

but added specific fractions of S

cells.

• Result - only DNA transformed

R cells into S cells.

• Result - not believed.

Hershey & Chase -1952

• Genetic information of a virus or

phage.

• Phage - virus that attacks

bacteria and reprograms host to

produce more viruses.

Bacteria with Phages

Phage Components

• Two main chemicals:• Protein

• DNA

• Which material is transferred to

the host?

Used Tracers

• Protein - CHONS, can trace

with 35S.

• DNA - CHONP, can trace with 32P.

Experiment

• Used phages labeled with one

tracer or the other and looked to

see which tracer entered the

bacteria cells.

Result

• DNA enters the host cell, but

the protein did not.

• Therefore:

DNA is the genetic material.

Picture Proof

Chargaff - 1947

• Studied the chemical

composition of DNA.

• Found that the nucleotides were

in certain ratios.

Chargaff’s Rule

• A = T

• G = C

• Example: in humans,A = 30.9%

T = 29.4%

G = 19.9%

C = 19.8%

Why?

• Not known until Watson and

Crick worked out the structure

of DNA.

Watson and Crick - 1953

• Used X-ray crystallography data

(from Rosalind Franklin)

• Used model building.

• Result - Double Helix Model of

DNA structure.

• (One page paper, 1953).

Rosalind Franklin

Book & Movies

• “The Double Helix” by James

Watson- His account of the

discovery of the shape of DNA

• Movie – The Double Helix

DNA Composition

• Deoxyribose Sugar (5-C)

• Phosphate

• Nitrogen Bases:• Purines

• Pyrimidines

DNA Backbone

• Polymer of sugar-phosphate.

• 2 backbones present.

Nitrogen Bases

• Bridge the backbones together.

• Purine + Pyrimidine = 3 rings.

• Constant distance between the

2 backbones.

• Held together by H-bonds.

Chargaff’s Rule

• Explained by double helix

model.

• A = T, 3 ring distance.

• G = C, 3 ring distance.

Watson and Crick

• Published a second paper

(1954) that speculated on the

way DNA replicates.

• Proof of replication given by

others.

Replication

• The process of making more

DNA from DNA.

• Problem: when cells replicate,

the genome must be copied

exactly.

• How is this done?

Models for DNA Replication

• Conservative - one old strand,

one new strand.

• Semiconservative - each strand

is 1/2 old, 1/2 new.

• Dispersive - strands are

mixtures of old and new.

Replication Models

Meselson – Stahl, late 1950’s

• Grew bacteria on two isotopes

of N.

• Started on 15N, switched to 14N.

• Looked at weight of DNA after

one, then 2 rounds of

replication.

Results

• Confirmed the

Semiconservative Model of DNA

replication.

Replication - Preview

• DNA splits by breaking the H-

bonds between the backbones.

• Then DNA builds the missing

backbone using the bases on

the old backbone as a template.

Origins of Replication

• Specific sites on the DNA

molecule that starts replication.

• Recognized by a specific DNA

base sequence.

Prokaryotic

• Circular DNA.

• 1 origin site.

• Replication runs in both

directions from the origin site.

Eukaryotic Cells

• Many origin sites.

• Replication bubbles fuse to form

new DNA strands.

DNA Elongation

• By DNA Polymerases such as

DNA pol III

• Adds DNA triphosphate

monomers to the growing

replication strand.

• Matches A to T and G to C.

Energy for Replication

• From the triphosphate

monomers.

• Loses two phosphates as each

monomer is added.

Problem of Antiparallel DNA

• The two DNA strands run

antiparallel to each other.

• DNA can only elongate in the

5’--> 3’ direction.

Leading Strand

• Continuous replication toward

the replication fork in the 5’-->3’

direction.

Leading Strand

• 1. DNA helicase unwinds the

DNA at the replication forks.• -leading strand (3’-5’), replicates (5’-3’)

towards the fork.

2. Molecules of single strand binding

protein prevent the DNA from sticking

back together.

3. Primase synthesizes an RNA primer at

the end of 5’end.

4. DNA pol III synthesizes the strand

continuously.

Lagging Strand

• 1. DNA helicase unwinds the

DNA at the replication fork.• -lagging strand (5’-3’) cannot replicate

in the 3’-5’ direction, replicates away

and towards the fork.

2. Primase joins RNA nucleotides into a

primer.

3. DNA pol III adds DNA nucleotides to

the primer forming an Okasaki fragment

1.

4. After reaching the next RNA primer

DNA pol III detaches.

• 5. Fragment 2 is primed, then

DNA pol III adds DNA

nucleotides, detaching when it

reaches the fragment 1 primer.

• 6. DNA pol I replaces the RNA

with DNA, adding nucleotides to

the 3’ end of fragment 2.

• 7. DNA ligase forms a bond

between the newest DNA and

the DNA of fragment 1.

• 8. This continues until the

strand is replicated.

Priming

• DNA pol III cannot initiate DNA

synthesis.

• Nucleotides can be added only

to an existing chain called a

Primer.

Primer

• Make of RNA.

• 10 nucleotides long.

• Added to DNA by an enzyme called Primase.

• DNA is then added to the RNA primer.

Priming

• A primer is needed for each

DNA elongation site.

Lagging Strand

• Discontinuous synthesis away

from the replication fork.

• Replicated in short segments as

more template becomes

opened up.

Okazaki Fragments

• Short segments (100-200

bases) that are made on the

lagging strand.

• All Okazaki fragments must be

primed.

• RNA primer is removed after

DNA is added.

Enzymes

• DNA pol I - replaces RNA

primers with DNA nucleotides.

• DNA Ligase - joins all DNA

fragments together.

Other Proteins in Replication

• Topoisomerase – relieves

strain ahead of replication

forks.

• Helicase - unwinds the DNA

double helix.

• Single-Strand Binding Proteins

- help hold the DNA strands

apart.

DNA Replication Error Rate

• 1 in 1 billion base pairs.

• About 3 mistakes in our DNA

each time it’s replicated.

Reasons for Accuracy

• DNA pol III self-checks and

corrects mismatches.

• DNA Repair Enzymes - a family

of enzymes that checks and

corrects DNA.

DNA Repair

• Over 130 different DNA repair

enzymes known.

• Failure to repair may lead to

Cancer or other health

problems.

Example:

• Xeroderma Pigmentosum -

Genetic condition where a DNA

repair enzyme doesn’t work.

• UV light causes damage, which

can lead to cancer.

Xeroderma Pigmentosum

Cancer Protected from UV

Thymine Dimers

• T-T binding from side to side

causing a bubble in DNA

backbone.

• Often caused by UV light.

Excision Repair

• Cuts out the damaged DNA.

• DNA Polymerase fills in the

excised area with new bases.

• DNA Ligase seals the

backbone.

Problem - ends of DNA

• DNA Polymerase can only add

nucleotides in the 5’--->3’

direction.

• It can’t complete the ends of the

DNA strand.

Result

• DNA gets shorter and shorter

with each round of replication.

Telomeres

• Repeating units of TTAGGG

(100- 1000 X) at the end of the

DNA strand (chromosome)

• Protects DNA from unwinding

and sticking together.

• Telomeres shorten with each

DNA replication.

Telomeres

Telomeres

• Serve as a “clock” to count how

many times DNA has replicated.

• When the telomeres are too short,

the cell dies by apoptosis.

Implication

• Telomeres are involved with the

aging process.

• Limits how many times a cell line

can divide.

Telomerase

• Enzyme that uses RNA to rebuild telomeres.

• Can make cells “immortal”.

• Found in cancer cells.

• Found in germ cells.

• Limited activity in active cells such as skin cells

Comment

• Control of Telomerase may stop

cancer, or extend the life span.

NEWS FLASH

• The DNA of Telomers is

actually used to build proteins.

• These proteins seem to impede

telomerase.

• Feedback Loop??