Molecular Genetics
Molecular Genetics
The Backstory
Mendel (1850’s)

Establishes rules of inheritance patterns
based on consistent experimental data.
Friedrich Miescher
1870’s
Isolates “nuclein”
from cells.
States function is to
store excess P for
the cell
WS Sutton (1903)

Establishes GENE-CHROMOSOME THEORY
identifies genes as the units found on
chromosomes, passed on to offspring
Phoebus Levine
c. 1915
makes distinctions
between amino
acids and
nucleotides within
cells
Levine’s Conclusion:
The code for inheritance lies in varying
sequences of amino acids!
The code for inheritance CANNOT come
from varying sequences of nucleotides!
Why such a conclusion?
>100,000 traits
20 amino acids
possible
combinations of
a.a. sequences to
code for traits!
Only 4 nucleotides!!
Not enough
combinations
possible to code for
the multitude of
traits found in
organisms!
Sounds good!
WRONG
…. But many accept Levine’s idea
Frederick Griffith
1928
Transformation
Experiment
Proves genetic
material can be
passed among
organisms, coding
for new phenotypes!
Mice
S. pneumoniae
Griffith’s Conclusion…
Something from heat killed type S is
transforming harmless type R into
harmful type S.
Type R now possesses the genetic code
required to produce a protective
capsule!
CRAZY!
Oswald Avery
1944
Rediscovers Griffith’s Transformation
Experiment
Concurs with Griffith that DNA is the
genetic material.. NOT protein!
Avery,McCleod, McCarty experiment:
STILL NOT ACCEPTED WIDELY!!
Hershey and Chase
1952
End debate over DNA vs. PROTEIN as
genetic material
E. coli / Bacteriophage (T2) experiment
T2 Bacteriophage
Erwin Chargaff
1947
Chargaff’s Findings:
DNA base composition varies among
species
Within a species, the amounts of
nitrogenous bases are present in a
definitive ratio!
Chargaff’s Rule:
In DNA, the amount of
Adenine = Thymine
Guanine = Cytosine
R. Franklin
M. Wilkins
J. Watson and
F. Crick
use Franklin’s crystallography photo to
determine the structure of DNA!!
double helix in structure
distance of .34 nm between adjacent
nucleotides
each strand 1nm wide
3.4 nm per turn of helix
L. Pauling
DNA STRUCTURE
Polymer of nucleotides



Deoxyribose (5 C sugar)
Phosphate (attached to 5` Carbon)
Nitrogenous base (attached to 1` Carbon)
Covalent bonding between adjacent
nucleotides !
(between 3`C of one sugar and the
5` phosphate of adjacent sugar)
3`, 5` phosphodiester bond
DNA strands are COMPLEMENTARY

The sequence of nucleotides in one strand
dictates the sequence in the other!
Bonding Between Complementary
Strands
held together by weak Hydrogen bonds
(responsbile for “double helix”
configuration!)
A always with T
Chargaff’s Rule
G always with C
Orientation of Complementary
Strands
ANTIPARALLEL arrangement!
one strand begins with a P group
attached to the 5`C of sugar and ends
where the P of the next nucleotide
would attach (ie: the 3`C)
adjacent strand is oriented in the
opposite way. (ie: it begins with 3`C
and ends with P on 5` C)
DNA is a double helix
P
T
A
5’
C
G
P
3’
P
DNA has directionality.
PP
P
Two nucleotide chains
together wind into a helix.
G
P
C
A
T
C
P
P
A sugar and phosphate
“backbone” connects
nucleotides in a chain.
P
G
Hydrogen bonds between
paired bases hold the two
DNA strands together.
P
P
3’
C
G
P
DNA strands are antiparallel.
5’
Orientation of
DNA
The carbon atoms on the sugar ring are numbered for
reference. The 5’ and 3’ hydroxyl groups (highlighted
on the left) are used to attach phosphate groups.
The directionality of a DNA strand is due to the
orientation of the phosphate-sugar backbone.
Structure of DNA.
1. Two nucleic acid chains running in opposite directions
2. The two nucleic acid chains are coiled around a central axis to
form a double helix
3. For each chain – the backbone comes from linking the
pentose sugar bases between nucleotides via
phosphodiester bonds connecting via 3’ to 5’
4. The bases face inward and pair in a highly specific fashion
with bases in the other chain
A only with T, G only with C
5. Because of this pairing, each strand is complementary to the
other
5’ ACGTC 3’
3’ TGCAG 5’
Thus DNA is double stranded
A gene: molecular definition
- A gene is a segment of DNA
- which directs the formation of RNA
- which in turn directs formation of a protein.
The protein (or functional RNA) creates the phenotype.
Information is conveyed by the sequence of the nucleotides.
Chromatin = DNA and associated
proteins
DNA winds around
histone proteins
(nucleosomes).
Other proteins wind
DNA into more tightly
packed form, the
chromosome.
Unwinding portions of
the chromosome is
important for mitosis,
replication and making
RNA.
Why is DNA a good material for storing genetic information?
A linear sequence of bases has a high storage capacity
a molecule of n bases has 4n combinations
just 10 nucleotides long -- 410 or
1,048,576 combinations
Humans – 3.2 x 109 nucleotides long – 3 billion base pairs
How do we know that DNA is the genetic material?
“A genetic material must carry out two jobs: duplicate itself
and control the development of the rest of the cell in a specific
way.”
-Francis Crick
Required properties of a genetic material
- Chromosomal localization
- Control protein synthesis
- Replication
DNA REPLICATION
Topic 3
DNA Replication
- the process of making new copies of the DNA molecules
Potential mechanisms:
organization of DNA strands
Conservative
old/old + new/new
Semiconservative
old/new + new/old
Dispersive
mixed old and new on each
strand
Meselson and Stahl’s replication
experiment
Conclusion: Replication is semiconservative.
Replication as a process
Double-stranded DNA unwinds.
The junction of the unwound
molecules is a replication fork.
A new strand is formed by pairing
complementary bases with the
old strand.
Two molecules are made.
Each has one new and one old
DNA strand.
Replication requires the coordinated regulation of many
enzymes and processes
- unwind the DNA
- synthesize a new nucleic acid polymer
- proof read
- repair mistakes
Fig 8.14
Enzymes in DNA
replication
Helicase unwinds
parental double helix
TOPOISOMERASE
TOO!
DNA polymerase
binds nucleotides
to form new strands
Binding proteins
stabilize separate
strands
Exonuclease removes
RNA primer and inserts
the correct bases
Primase adds
short primer
to template strand
Ligase joins Okazaki
fragments and seals
other nicks in sugarphosphate backbone
Replication
3’
3’
5’
5’
3’
5’
3’
5’
Helicase and Topoisomerase proteins bind to DNA
sequences and unwinds DNA strands, breaking H bonds
between base pairs.
Binding proteins prevent single strands from rewinding.
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.
Replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
3’
5’
DNA polymeraseIII enzyme adds DNA nucleotides
to the RNA primer.
Replication
Overall direction
of replication
3’
5’
3’
5’
3’
5’
3’
5’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
Replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
3’
5’
Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
5’
3’ 5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
5’
3’ 5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
DNA Synthesis
•Synthesis on leading and lagging strands
•Proofreading and error correction during DNA replication
•Simultaneous replication
occurs via looping of lagging
strand
Replication
3’
5’
3’
5’
3’
5’
3’ 5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Exonuclease enzymes remove RNA primers.
Replication
3’
3’
5’
3’
5’
3’5’
3’
5’
Exonuclease enzymes remove RNA primers.
Ligase forms bonds between sugar-phosphate
backbone.
http://www.stolaf.edu/people/giannini/f
lashanimat/molgenetics/dna-rna2.swf
DNA Replication Overview
Topoisomerase
Helicase
DNA Polymerase
Direction of Synthesis? The Big
Prob?
DNA ligase
Repair Nuclease
Role of RNA in DNA replication?
The Mutation Repair Issue
Ultimate Error Rate:
~ 1 / 1,000,000,000 nucleotides
Initial Error Rate:
~ 1 / 10,000 nucleotides
First line of Defense: Polymerase
(mismatch repair)
Second Line:
NUCLEASE !
Nuclease functions to…
Repair mutations due to
environmental mutagen
exposure !
(EXISION REPAIR)
The most common mutagen…
UV radiation exposure !
Thymine-Dimer formation!
Disorted DNA molecule!
Prevents future DNA replication!
Big Problem!
Exision Repair Process
Nuclease cuts damaged DNA at two
points
DNA polymerase fills the gap with
undamaged nucleotides
DNA ligase seals phosphodiester
bonds
All done.