DNA Replication
[Link]
Today’s Agenda
Introduction to the central dogma
History
Introduction to DNA
DNA replication
Central Dogma of Biology
[Link]
History
Gregor Mendel and his pea
plants (1856)
Deemed “Father of genetics”
Used pea plants to discover
”dominant
and “recessive” traits
Determined that traits were
inherited via genes passed
from parents to offspring
Traits are inherited
independently
[Link]
History
Thomas Hunt Morgan
and his fruit flies
Determined that genes
were carried on
chromosomes
Followed fly eye color
mutations
[Link]
lassical-genetics/chromosomal-basis-of-
genetics/a/discovery-of-the-chromosomal-basis-
of-inheritance
History
Frederick Griffith and his
bacteria (1928)
Determined that genetic
material is transferrable
Used nonvirulent “R” strain
and virulent “S” strain of
bacteria
Virulent = disease causing
Injected into mice
[Link]
Conclusion: R- strain bacteria were “transformed” with something
from the dead S- strain that caused them to become virulent
History
Hershey Chase
Experiment (1952)
Alfred Hershey and
Martha Chase
Determined that
genetic material is
DNA
Used radioactive
bacteriophages
(viruses that infect
[Link]
bacteria)
Phosphorus: incorporated into
DNA
Sulfur: incorporated into protein
Breakout Rooms (~ 5 min)
Rosalind Franklin, James
Watson, and Francis
Crick (1953)
What did they discover?
What methods did they
use?
DNA: Deoxyribonucleic acid
Nucleotide
components:
1. Phosphate
2. Sugar
3. Nitrogenous base
[Link]
Bases and
Base Pairing
• Two types of bases
• Pyrimidines
• 6-membered ring
• Cytosine and
Thymine
• Purines
• 6-membered ring
attached to 5-
membered ring
• Adenine and Guanine
• Base pairing
• Adenine – thymine
• Cytosine – Guanine
[Link]
rsch_et_al.)/28%3A_Biomolecules_-_Nucleic_Acids
DNA Structure
DNA Structure
Nucleotides bind together
and become polymers via
phosphodiester bond
Phosphate group binds to
3’ carbon of the preceding
base to the 5’ carbon of the
next base
Called the phosphodiester
bone
Makes up the phosphate-
sugar backbone
DNA Structure
DNA strands are anti-
parallel to one another
The strands run in
opposite direction
The forward strand runs
from 5’ to 3’
The reverse strand runs
from 3’ to 5’
Refers to the carbon in
which the phosphate is
attached to
[Link]
ral_Biology_l_-_Laboratory_Manual/01%3A_Labs/1.10%3A_DNA_
DNA Structure
Breakout Rooms (~ 7 min)
What is a nucleotide? What does it consist of?
What connects bases on the same DNA strand to
each other?
What holds bases on different DNA strands
together?
What are the base pairing rules for DNA?
Which is the complementary
strand?
5’ ATTGCACCCGT 3'
a. 5’ TAACGTGGGCA 3’
b. 5’ ACGGGTGCAAT 3’
c. 5’ ATTGCACCCGT 3’
d. 5’ TGCCCACGTTA 3’
e. None of the above
If a segment of DNA has 22%
composition of guanine
molecules, what is the
composition percentage of
adenine?
a. 22%
b. 78%
c. 28%
d. 56%
[Link] of the above
10 min break
DNA Replication
Occurs prior to cell division, in
the S phase of the cell cycle
Semiconservative
Half of the parental DNA goes
into the new DNA
New DNA has 1 new strand and
1 old strand
[Link]
semiconservative-and-dispersive-replication/
[Link]
DNA Replication Fork
Mechanism of Replication
1. Initiation
1. Preinitiation complex
2. Unwinding of the DNA at the
origin of replication
2. Elongation
1. RNA primers prime the new
strand
2. The new daughter strand is
built complementary to the
parental strand
1. Always 5’ to 3’!!
3. Termination
1. Termination of the replication
fork
[Link]
Main players
Helicase Unwinds the DNA
RNA primase Makes short complementary RNA
primers
DNA polymerase III Adds DNA nucleotides to the 3’ end
of the new strand
DNA polymerase I Fills in the RNA primer gaps with
DNA
RNase and other nucleases Cleaves and degrades RNA primers
DNA ligase Joins the Okazaki fragments
together into one strand
Telomerase Extends region of the telomere
Initiation
DNA needs to be un-winded so that the DNA
strands are accessible
DNA Helicase – breaks H bonds and
unwinds and unzips DNA helix
Topoisomerase (DNA Gyrase) – relieves
supercoiling downstream
SSB – keeps strands separated
Complementary RNA primers made by
primase anneal to the parental DNA
This is because DNA polymerase can
only be initiated with a free 3’ end
Elongation
Main player: DNA
polymerase
Starts synthesizing from
RNA primers
Protein that adds
nucleotides to complement
the parent strand
Can only synthesis in the
5’à3’ direction
Leading strand synthesis
Synthesized continuously as
helicase unwinds the DNA
Only 1 RNA primer needed
Lagging strand synthesis
Synthesized in pieces
(Okazaki fragments)
Requires lots of RNA primers
[Link]
14668888/
[Link]
Semidiscontinous Replication
DNA Pol I
DNA
ligase
DNA Pol
III
Termination
DNA synthesis happens
with multiple origins of
replication
One replication fork will
reach the other
DNA polymerase is then
blocked by another new
synthesized strand of DNA
and falls off
RNase H and
endonucleases then remove
all the RNA primers
DNA polymerase I fills in
the gaps
DNA ligase joins the DNA
strands together
[Link]
End Replication Problem
If DNA polymerase can
only add nucleotides to
an existing nucleic acid,
then what happens at
the site of the very first
RNA primer?
The last Okazaki
fragment can’t be
replaced
with every round of
replication the
chromosome should get
shorter :O [Link]
replication/a/telomeres-telomerase
Telomerase and Telomeres
Telomeres are found at the ends
of our chromosomes
Repetitive regions
Hundreds or thousands of 5'-
TTAGGG-3’ repeats
Shorten with every cell division
Telomerase can reverse telomere
shortening by adding these
repeats
Only makes 1 strand, primase and
DNA polymerase makes the other
Active in germ cells but not
somatic cells
Telomerase is often upregulated
in cancer
Why?
[Link]
genetic-material/dna-replication/a/telomeres-telomerase
Which segment is synthesized
first?
a.
b.
c.
d.
e.
f.
DNA Repair
Damaged via chemical or UV light
UV light causes thymine dimers
Photorepair via enzymes called
Photolyase cleaves dimers
Different types of damage and repair
Proofreading by DNA polymerase
Mismatch excision repair pathway
Base excision repair pathway
Nucleotide excision repair pathway
Homologous recombination
Repair by end joining
PARP inhibitors in BRCA1
mutant breast cancer
PARP-1- DNA repair enzyme
Poly ADP ribose polymerase
Recognizes single strand breaks and recruits repair
enzymes
BRCA1 and BRCA2
Repairs double stranded DNA breaks
People with mutant BRCAs are more likely to develop
breast cancer, ovarian cancer, or prostate cancer
More mutations accumulate
PARP inhibitors causes cell death
Accelerates double stranded breaks
Very effective in BRCA mutant -/- cells
Proofreading Function of DNA Polymerase