DNA and
the Gene:
Synthesis and Repair
Chapter 15
Outline
DNA structure
DNA synthesis
DNA repair
Are genes made of DNA or protein?
Proteins:
More complex
More variable
DNA:
Only four different
nucleotides
Only the genes of a virus enter a
host cell
Does protein of DNA enter the cell?
Radioactive isotopes can tag DNA
and protein
Allow virus to
infect E.coli
Collect cell
cultures
Results: Genes consists of DNA
Which of the following best describes the composition of
DNA monomers?
A. All of the monomers contain ribose connected to
one of four nitrogenous bases.
B. All of the monomers contain a phosphate
group connected to a nitrogenous base.
C. All of the monomers contain a phosphate group
and a nitrogenous base connected to deoxyribose.
D. All of the monomers contain a cytosine
base connected to a deoxyribose.
Structure of DNA
Structure of DNA
Structure of DNA
5' end = phosphate
3' end = hydroxyl group
Complementary
base pairing and the
DNA double helix
Complementary
base pairing and the
DNA double helix
Guanine (G) hydrogen-
bonds with cytosine (C)
Adenine (A) hydrogen-
bonds with thymine (T)
Stabilized with
interactions between
stacked base pairs
The secondary structure of a double-stranded DNA helix
molecule can best be described as a _____.
A. sugar-phosphate backbone with the bases of the complementary base pairs
adenine-thymine and guanine-cytosine projecting toward the inside of the
backbone
B. sugar-phosphate backbone with adenine, thymine, guanine, or cytosine
bases projecting toward the outside of the backbone
C. sugar-phosphate backbone that is made up of adenine, thymine, guanine, or
cytosine bases hydrogen-bonded to each other through phosphodiester bonds
D. sugar-phosphate backbone with the complementary base pairs
adenine-guanine and thymine-cytosine joined by hydrogen bonding
HOW IS DNA SYNTHESIZED?
1.SEMICONSERVATIVE
REPLICATION
2. CONSERVATIVE
REPLICATION
3. DISPERSIVE REPLICATION
DNA replication is semiconservative
because replicated DNA molecules are
composed of _____.
A. two strands of old DNA
B. two strands of new DNA
C. one strand of old DNA and one strand of new DNA
D. one strand of new DNA
THE MESELSON-STAHL EXPERIMENT
N15/N14 isotopes to label DNA
Bacteria copy their entire genome in cell division
THE MESELSON-STAHL EXPERIMENT
What is the difference between isotope N15 and N14?
SEMICONSERVATIVE REPLICATION
Parental strands
separate, and each is
template for new
daughter strand:
Each daughter has one
old and one new strand
CONSERVATIVE REPLICATION
Parental molecule
serves as an entirely
new molecule:
One daughter has both
old strands; other has
both new strands
DISPERSIVE REPLICATION
Parent molecule is cut
into small pieces
Each daughter has old
and new DN A
interspersed
RESULTS
RESULTS
DNA polymerase catalyzes DNA
synthesis
Several types of DNA Polymerases
Work only in one direction
Can add deoxyribonucleotides only to 3' end of a
growing DNA chain
DNA SYNTHESIZED ALWAYS PROCEEDS 5' --> 3'
DIRECTION
DNA Synthesis only goes 5' to 3'
ENERGY OF DNA SYNTHESIS
Endergonic:
Requires input of energy
Monomer = dNTPs
deoxyribonucleoside
triphosphates
High potential energy due to
phosphate groups
Enough energy to create
phosphodiester bonds exergonic
WHERE DOES REPLICATION START?
REPLICATION STARTS AT ORIGIN OF REPLICATION
REPLICATION STARTS AT ORIGIN OF REPLICATION
REPLICATION STARTS AT ORIGIN OF REPLICATION
Bacteria have one replication bubble
Eukaryotic cells have many on each
chromosome
Each replication bubble has two replication
forks
Synthesis is bidirectional
Replication bubbles grow in both directions
The following DNA sequence 3'AATTGCAGATTCA 5' occurs
at a DNA origin of replication. Which of the sequences
below would most likely bind to this DNA sequence as a
primer to initiate replication?
A. 3' TTAACGTCTAA 5'
B. 5' TTAACGTCTAA 3'
C. 5' UUAACGUCUAA 3'
D. 3' UUAACGUCUAA 5'
Cytosine makes up 42% of the nucleotides in a sample of
DNA from an organism. Approximately what percentage
of the nucleotides in this sample will be thymine?
A. 16%
B. 58%
C. 8%
D. 42%
In analyzing the number of different bases in a DNA
sample, which result would be consistent with the base-
pairing rules?
A. A + T = G + C
B. A + G = C + T
C. A = C
D. A = G
HOW IS THE HELIX OPENED AND STABILIZED?
DNA helicase:
Breaks hydrogen bonds between DNA strands
Single-strand DNA-binding proteins (SSBPs):
Attach to separated strands to prevent them from closing
Topoisomerase:
Cuts and rejoins DNA to relieve tension caused by unwinding
DNA
LEADING-STRAND SYNTHESIS
DNA POLYMERASE
Can only synthesize 5' --> 3'
Cannot start synthesis from scratch on template strand
Can only extend from 3' end of existing strand
PRIMASE CREATES PRIMER
Type of RNA polymerase
Does not require free 3' end to begin synthesis
Base pairs to DNA template
DNA and RNA can be a primer
Supplies 3' end for DNA polymerase
LEADING-STRAND SYNTHESIS
Which of the following sequences would result
from replication of this DNA template strand?
3′-A A G T C A G T-5′
A. 5′-A A G T C A G T-3′
B. 5′-T T C A G T C A-3′
C. 3′-T T C A G T C A-5′
D. 3′-A A G T C A G T-5′
The leading and lagging strand
Lagging strand synthesis
Lagging strand synthesis
Okazaki fragment
1960s, Reiji and Tsuneko
Okazaki
Discovered short strands
attached to primers
Fragments are linked
together after being
formed
Lagging strand is
synthesized as short
discontinuous Okazaki
gragments
Lagging strand synthesis
Lagging strand synthesis
Lagging strand synthesis
Lagging strand
synthesis
Primase synthesizes new
RNA primers on lagging
strand as replication fork
opens
DNA polymerase
synthesizes short
fragments of D NA along
lagging strand
Fragments are then linked
into continuous strand
The leading and lagging strand
Draw out the replication bubble
The leading and lagging strand
Opening the helix:
Leading-strand synthesis
Lagging-strand synthesis
Which enzyme is incorrectly
paired with its function?
A. DNA polymerase III—proofread DNA base pairs
B. DNA ligase—synthesize Okazaki fragments
C. DNA helicase—unwind DNA
D. primase—creates RNA primer
DNA polymerase cannot replicate DNA unless an RNA
primer is first attached to the template strand. This is
because DNA polymerase can only _____.
A. add onto existing 3′ hydroxyl groups
B. replicate in a 5′ → 3′ direction
C. replicate the leading strand
D. add onto ribonucleotides
THE REPLISOME IS A DNA SYNTHESIZING MACHINE
Replisome is dynamic
Continuous strand
may not be
synthesized as
continuously as once
believed
Rates of DNA
synthesis may vary
widely
REPLICATING THE ENDS OF LINEAR CHROMOSOMES
REPLICATING THE ENDS OF LINEAR CHROMOSOMES
REPLICATING THE ENDS OF LINEAR CHROMOSOMES
REPLICATING THE ENDS OF LINEAR CHROMOSOMES
Single strand DNA is degraded
TELOMERASE PREVENTS SHORTENING OF
TELOMERES DURING REPLICATION
Which of the following statements is true of DNA
synthesis?
A. DNA polymerases add nucleotides in the 3' → 5'
direction to complete the complementary strand.
B. DNA synthesis proceeds in one direction from
a point of origin.
C. DNA polymerase requires a single-stranded template in addition to
the short complementary RNA primer in order to synthesize DNA.
D. DNA polymerases catalyze the random addition
of monomers to extend the DNA polymer chain.
TELOMERASE PREVENTS SHORTENING OF
TELOMERES DURING REPLICATION
TELOMERASE PREVENTS SHORTENING OF
TELOMERES DURING REPLICATION
TELOMERASE PREVENTS SHORTENING OF
TELOMERES DURING REPLICATION
TELOMERASE PREVENTS SHORTENING OF
TELOMERES DURING REPLICATION
TELOMERASE PREVENTS SHORTENING OF
TELOMERES DURING REPLICATION
1. The 3' end of the lagging strand forms single-
strand “overhang”
2. Telomerase binds to overhang and uses RNA that
it carries as template for DNA snythesis
3. Telomerase continues to move down new strand,
adding more short DNA sequences to the end
4. Once overhang is long enough, normal DNA
synthesis can occur
TELOMERES
Telomeres do not
contain genes
Consist of short,
repeating stretches
of bases
EFFECT OF TELOMERE LENGTH ON CELL DIVISION
Telomerase primarily found in gametes
and stem cells
Somatic cells normally lack telomerase:
Chromosomes progressively shorten
as individual ages
EFFECT OF TELOMERE LENGTH ON CELL DIVISION
Once chromosome are shortened to
threshold length, further divisions are
shut down
In culture, telomere length correlates to
the number of cell divisions
EFFECT OF TELOMERE LENGTH ON CELL DIVISION
What would happen if you added
telomerase to cell cultures?
EFFECT OF TELOMERE LENGTH ON CELL DIVISION
What would happen if you added
telomerase to cell cultures?
Allows them to continue to divide
Cancer usually has active
telomerase
Allows unlimited cell division
Correcting mistakes in
DNA synthesis
DNA replication is very accurate
DNA polymerase matches bases with high accuracy:
Correct bases are the most energetically favorable
Correct base pairs have a distinct shape
Inserts an incorrect base about once every 100,000
bases
Repair enzymes remove defective bases and replace
them with the correct one
Correcting mistakes in
DNA synthesis
D N A polymerase work fast but we need
accuracy as well:
Overall error rate during replication is one
error per billion deoxyribonucleotides
Even though errors are rare, they are major
contributors to development of cancer
Accurate DNA replication is a matter of life
and death
Correcting mistakes in
DNA synthesis
DNA replication is very accurate
DNA polymerase matches bases with high accuracy:
Correct bases are the most energetically favorable
Correct base pairs have a distinct shape
Inserts an incorrect base about once every 100,000
bases
Repair enzymes remove defective bases and replace
them with the correct one
DNA polymerase can proofread
Mismatched bases have distinct shape
D N A polymerase will add nucleotide only if previous
base pair is correct
DNA polymerase can proofread
Exonuclease active site:
Site catalyzes removal of incorrect deoxyribonucleotide
DNA polymerase can proofread
Inserts correct nucleotide
DNA polymerase can proofread
DNA polymerase can proofread
D N A polymerase sometimes leaves a mismatched pair
behind
Mismatch repair occurs when mismatched bases are
corrected after D N A synthesis is complete
Mismatch repair enzymes:
Recognize mismatched pair
Remove section of newly synthesized strand that
contains incorrect base
Fills in correct bases
UV light damages DNA
Kink blocks DNA
replication
Nucleotide excision repair
Nucleotide excision repair
Nucleotide excision repair
Nucleotide excision repair
Nucleotide excision repair
Nucleotide excision repair
Protein complex recognizes kink
Removes damaged single-
stranded DNA
Uses intact strand as template
for new DNA
DNA ligase links repaired strand
to original undamaged DNA
Xeroderma pigmentosum (XP)
Rare autosomal recessive disease in humans
Causes extreme sensitivity to UV light
Increases chance of skin cancer by 1000-2000 times
XP caused by mutations in nucleotide excision repair
systems:
Cannot repair DNA damaged by ultraviolet radiation
Can result from mutations in any of eight genes
Mutations in genes required for DNA repair
are frequently associated with cancer
If mutations in genes involved in cell cycle go unrepaired:
Cell may begin to grow in uncontrolled manner
This growth can result in formation of a tumor
If overall mutation rate in a cell is elevated:
Because of mutations in DNA repair genes, the
mutations that trigger cancer become more likely
Benzopyrene in cigarette smoke binds to DNA and
distorts its shape, interfering with DNA replication. Which
repair mechanism would most likely be used to repair
the damage caused by this chemical?
A. proofreading
B. mismatch repair
C. telomere repair
D. nucleotide excision repair
Homework:
Chapter 15 due Nov 15 @ midnight
Exam 3 November 13th
