Download Chapter 16: DNA Structure & Replication 1. DNA Structure 2. DNA Replication

Chapter 16:
DNA Structure & Replication
1. DNA Structure
2. DNA Replication
1. DNA Structure
Chapter Reading – pp. 313-318
Genetic Material: Protein or DNA?
Until the early 1950’s no one knew for sure, but it
was generally thought that protein was the genetic
material. Why?
• protein is made of 20 different amino acids
• DNA is made of only 4 different nucleotides
• protein could theoretically store more info
• a “20 letter alphabet” vs a “4 letter alphabet”
• it was assumed that life was so complex, therefore a
“bigger alphabet” was necessary to somehow encode it!
Some classic experiments would prove otherwise…
Transformation of Bacteria
EXPERIMENT
Living S cells
(control)
Living R cells
(control)
Heat-killed
S cells
(control)
Mixture of
heat-killed
S cells and
living R cells
Demonstrated
the transfer of
a genetic trait
between
different
bacteria.
The nature of
that genetic
material was
still unknown.
RESULTS
Mouse dies
Mouse healthy Mouse healthy Mouse dies
(Frederick Griffith, 1928)
Living S cells
What is the Genetic Material of
Bacteriophages?
Bacteriophages are viruses that infect bacteria.
• consist of a protein capsid which contains DNA
What enters the
bacterial host
cell, viral protein,
DNA, or both?
Tail
sheath
Tail fiber
DNA
Bacterial
cell
100 nm
• whatever enters
the host cell
should be the
genetic material
Phage
head
Bacteriophage Genetic Material is DNA
EXPERIMENT
Phage
Radioactive
protein
Empty
protein
shell
Radioactivity
(phage protein)
in liquid
Bacterial cell
Batch 1:
Radioactive
sulfur
(35S)
DNA
Phage
DNA
Centrifuge
Pellet (bacterial
cells and contents)
Radioactive
DNA
Batch 2:
Radioactive
phosphorus
(32P)
Centrifuge
(Alfred Hershey & Martha Chase,
Radioactivity
Pellet (phage DNA)
in pellet
1952)
The Discovery of DNA Structure
Using the technique of x-ray crystallography,
Rosalind Franklin, James Watson & Francis Crick
figured out the structure of DNA
• Watson & Crick used the X-ray diffraction data of Rosalind
Franklin to deduce the structure of DNA
(a) Rosalind Franklin
(b) Franklin’s X-ray diffraction
photograph of DNA
DNA: a Polymer of 4 Nucleotides
pyrimidines
purines
A
T
C
G
Sugar–phosphate
backbone
Nitrogenous bases
5 end
Thymine (T)
Adenine (A)
Cytosine (C)
Phosphate
Guanine (G)
Sugar
(deoxyribose)
DNA
nucleotide
3 end
Nitrogenous base
Structure of a
DNA Strand
A single DNA polymer
or “strand” consists of
a sugar-phosphate
backbone with the
bases project out.
The ends of a DNA
strand are different,
with one end having a
free 5’ phosphate, and
the other having a free
3’ hydroxyl group
Structure of Double-stranded DNA
• the 2 strands are anti-parallel and interact via base pairs
C
5 end
G
C
Hydrogen bond
G
C
G
3 end
C
G
A
T
3.4 nm
A
T
C
G
C
G
A
T
1 nm
C
A
G
C
G
A
G
A
T
3 end
T
A
T
G
C
T
C
C
G
T
0.34 nm
5 end
A
(a) Key features of
DNA structure
(b) Partial chemical structure
(c) Space-filling
model
DNA “Base-Pairing”
Base pairs are held together by hydrogen bonds.
Why only A:T and C:G?
• the position of chemical
groups involved in H-Bonds
Sugar
Sugar
Adenine (A)
Thymine (T)
• the size of the bases
(purine & pyrimidine)
Purine  purine: too wide
Sugar
Pyrimidine  pyrimidine: too narrow
Sugar
Guanine (G)
Cytosine (C)
Purine  pyrimidine: width
consistent with X-ray data
The DNA “Sequence”
The DNA sequence is the linear order of
nucleotides in a DNA strand:
• each DNA strand in the double helix has its own
sequence
• the sequences in each strand are considered as
complementary to each other
• they differ, but “fit just right” with each other
• ea strand will “fit” with only 1 complementary strand
e.g.
5’
3’
A–C–A–C–A–C–A–C–A–C 3’
T–G–T–G–T–G–T–G–T–G 5’
2. DNA Replication
Chapter Reading – pp. 318-330
How is DNA Replicated?
Every time a cell reproduces (i.e., divides) it
must replicate its chromosomes (DNA) during
S phase.
The process of DNA replication was originally
proposed to depend on the rules of base pairing:
• A:T & T:A , C:G & G:C
• the sequence of one strand dictates the
sequence of the other
• each strand of the double helix could serve as a
template to make a complementary strand
Model for DNA Replication
A
T
A
T
A
T
A
T
C
G
C
G
C
G
C
G
T
A
T
A
T
A
T
A
A
T
A
T
A
T
A
T
G
C
G
C
G
C
G
C
(a) Parent molecule
(b) Separation of
strands
(c) “Daughter” DNA molecules,
each consisting of one
parental strand and one
new strand
The semiconservative model of DNA replication
proposed that each original strand serves as a template
to produce a new complementary strand.
• note that ea original strand ends up in a different molecule
Parent
cell
(a) Conservative
model
First
Second
replication replication
Other
Models
CONSERVATIVE
The original DNA
strands stay together
(b) Semiconservative
model
SEMICONSERVATIVE
The original DNA
strands remain intact
in separate molecules
(c) Dispersive model
DISPERSIVE
The original DNA
strands are dispersed
among the all daughter
strands
Testing the
Models
In this experiment,
bacteria with DNA
containing the “heavy”
isotope 15N were
allowed to reproduce
in medium containing
lighter 14N.
Density-gradient
centrifugation revealed
that DNA replication is
semiconservative.
Matthew Meselson &
Franklin Stahl, 1958
EXPERIMENT
1 Bacteria
cultured in
medium with
15N (heavy
isotope)
2 Bacteria
transferred to
medium with
14N (lighter
isotope)
RESULTS
3 DNA sample
centrifuged
after first
replication
CONCLUSION
Predictions:
First replication
Conservative
model
Semiconservative
model
Dispersive
model
4 DNA sample
centrifuged
after second
replication
Less
dense
More
dense
Second replication
DNA Replication in Bacteria
(a) Origin of replication in an E. coli cell
Origin of
replication
Parental (template) strand
Daughter (new) strand
Doublestranded
DNA molecule
Replication
bubble
Initiation of DNA
replication
requires an origin
of replication
Replication fork
Two
daughter
DNA molecules
0.5 m
DNA Replication in Eukaryotes
(b) Origins of replication in a eukaryotic cell
Origin of replication
Parental (template)
strand
Bubble
Double-stranded
DNA molecule
Daughter (new)
strand
Eukaryotic DNA
replication
requires multiple
origins of
replication
Replication fork
Two daughter DNA molecules
0.25 m
Overview of DNA Replication
Leading
strand
Overview
Origin of replication
Lagging
strand
Primer
Leading
strand
Lagging
strand
Overall directions
of replication
DNA Replication proceeds 5’ to 3’
Template strand
3
New strand
5
Sugar
Phosphate
A
Base
T
C
G
G
C
DNA
polymerase
can add
only to the
3’ end
A
P
C
Nucleoside
triphosphate
3
A
T
C
G
G
C
T
A
DNA
polymerase
OH
3
5
OH
Pi
Pyrophosphate
3
C
2Pi
5
5
Enzymes involved in DNA Replication
DNA Polymerase – synthesizes new DNA
Helicase – unwinds DNA double helix
Topoisomerase – relieves tension due to DNA
unwinding
Primase
Topoisomerase
3
5
5
3
Primase –
3
makes short
RNA RNA primers
primer
Helicase
5
Single-strand binding
proteins
DNA Ligase –
connects DNA
fragments
Leading Strand DNA Synthesis
Proceeds toward unwinding replication fork:
Origin of
replication
3
5
RNA primer
5
3
3
Sliding clamp
DNA pol III
Parental DNA
5
3
5
5
3
3
5
DNA synthesis is
continuous on
the leading
strand only
DNA polymerase
can only
synthesize DNA
in a 5’ to 3’
direction.
DNA polymerase
requires an RNA
primer which it
can extend in a
continuous
manner toward
the unwinding
replication fork
Lagging Strand DNA Synthesis
3
5
Template
strand
Proceeds away from the
unwinding replication fork:
3
5
3
RNA primer
for fragment 1
5
1
• DNA polymerase
synthesizes DNA in
Okazaki fragments
3
5
Okazaki
fragment 1
3
5
RNA primer
for fragment 2
1
• each fragment requires
an RNA primer
3
5
5
3
2
Okazaki
fragment 2
1
5
3
DNA synthesis is
discontinuous
on the lagging
strand
• another DNA
polymerase will replace
the RNA with DNA
3
5
2
1
5
3
3
5
• DNA ligase will link the
fragments together
2
1
3
5
Overall direction of replication
Summary of DNA Replication
Overview
Origin of
replication
Leading
strand
Lagging
strand
Leading
strand
Lagging
strand
Overall directions
of replication
Leading strand
DNA pol III
5
3
3
Parental
DNA
Primer
5
3
Primase
5
DNA pol III
4
Lagging strand
DNA pol I
35
3
2
DNA ligase
1 3
5
DNA replication proceeds in this manner in ALL
living organisms.
Current Model of DNA Replication
DNA pol III
Parental DNA
5
3
5
3
5
5
Connecting
protein
3
Helicase
3
DNA
pol III
Leading strand
3
5
3
5
Lagging strand
Lagging
strand
template
5
3
3
5
Nuclease
5
3
3
5
DNA
polymerase
DNA Repair
When DNA is damaged it is
essential that the DNA is
repaired so it can be replicated
and expressed properly.
• special enzymes recognize and
remove the damaged portion of DNA
5
3
3
5
• a DNA polymerase will fill in the gap
5
3
3
5
• DNA ligase will then connect the
newly made DNA to the adjacent
strand
DNA
ligase
The Problem with Telomeres
The ends of linear
chromosomes, the
telomeres, cannot be
completely copied on
the lagging strand.
5
Leading strand
Lagging strand
Ends of parental
DNA strands
3
Last fragment
RNA primer
Lagging strand
5
3
Parental strand
This results in
progressive shortening
of the chromosome
every time it is
replicated.
Next-to-last fragment
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Second round
of replication
5
New leading strand 3
New lagging strand 5
Telomerase will solve
this problem in certain
cell types
3
Further rounds
of replication
Shorter and shorter daughter molecules
…more on Chromatin
Chromatin refers to the complex of DNA and
histone proteins in eukaryotic nuclei:
• chromosomal DNA wraps around histone
proteins to form structures called nucleosomes
that look like “beads on a string”
• different parts of a chromosome can be in
various states of “packing”
EUCHROMATIN – loosely packed DNA
HETEROCHROMATIN – tightly packed DNA
Key Terms for Chapter 16
• bacterial transformation, bacteriophage
• x-ray crystallography
• pyrimidine, purine, base-pair, complementary
• double helix, anti-parallel, sugar-phosphate
backbone
• DNA replication, template
• conservative, semiconservative, dispersive
• DNA polymerase, helicase, topoisomerase,
primase, DNA ligase
Relevant
Chapter
Questions
1-7, 9
• leading, lagging strand; continuous, discontinuous
• Okazaki fragment, telomere, telomerase
• chromatin, nucleosome, euchromatin, heterochromatin