DNA Synthesis Lecture

In the Hersey-Chase experiments, how did the
scientists show that the genetic material was DNA?

The P32-labeled viral DNA was in the cell pellet when they centrifuged down the cells.
Why can DNA strands only be synthesized in one direction?
The polymerization reaction involves an attack of the 3’OH on an incoming nucleotide
What did early researchers know about chromosomes?
That they consisted of DNA and protein
Why was the old general consensus that genes were made of proteins?
-The relative complexity and variability of proteins
– Comparison to DNA, which is comprised of only
four different nucleotides
Who conducted the first experiments to see if genes were DNA or protein?
Alfred Hershey and Martha Chase
What virus was studied by Hershey and Chase?
T2 (bacteriophage to E. coli)
What is the first step of T2 infecting E. coli?
Attaches to the cell and injects its genes into the cell
What is the T2 injected material considered and why?
It is considered genetic material because it directs the production of new virus particles
What is left behind during the T2 infection?
the protein coat, or capsid, of the
original parent virus is left behind as a ghost attached
to the exterior of the cell
What were the two radioactive isotopes in the Hershey/Chase experiment?
32P to label DNA or 35S to label proteins
What would happen in the H/C experiment if the genes consisted of DNA?
-Then 32P-labeled DNA would be found inside the
cells
– While 35S-labeled proteins would be found only
in the ghosts outside the cells
Which radioactive isotope was found in cell pellets? (H/C)
32P-labeled DNA
Which radioactive isotope was found in viral capsids (solution) (H/C)?
35S-labeled protein
What was the result of the Hershey/Chase experiment?
This result supports the proposal that genes are made of DNA, not protein.
What makes up the primary structure of a single strand of DNA?
1. A backbone of sugar (deoxyribose) and phosphate groups
2. Nitrogen-containing bases (A, C, G, T) that project from the backbone
3. The individual repeating units are called deoxyribonucleotides
Describe the directionality of DNA strands:
-One end has an exposed hydroxyl group on the 3′ carbon of deoxyribose
-The other end has an exposed phosphate group on a 5′ carbon
-The molecule thus has a 3′ end and a 5′ end
Who solved the secondary structure of DNA?
Watson and Crick (1953)
Watson and Crick proposal:
DNA is double stranded
-two DNA strands line up in the opposite direction to each other in antiparallel fashion
What does Adenine bind with?
thymine
What does Guanine bind with?
cytosine
What is the secondary structure of DNA stabilized by?
by base pairing involving hydrogen bonds
Watson and Crick proposal II:
-The existing strands of DNA served as a template (pattern) for the production of new strands
-Bases were added to the new strands according to complementary base pairing
Conservative replication:
The parental molecule serves as a template for the synthesis of an entirely new double stranded molecule
Semiconservative replication:
-The parental DNA strands separate
-Each is used as a template for the synthesis of a new daughter strand
-DNA molecules each consist of one old parental and one new daughter strand
Dispersive replication:
-The parent molecule is replicated in sections
-The replicated molecules contain old DNA interspersed with newly synthesized DNA
What does the polymerization reaction lead to?
the formation of a phosphodiester bond between polymerized nucleotides
Phosphodiester bond:
a chemical bond of the kind joining successive sugar molecules in a polynucleotide. (a phosphorus atom involved in two ester bonds.)
Polynucleotide:
a linear polymer whose molecule is composed of many nucleotide units, constituting a section of a nucleic acid molecule.
Where can DNA polymerases add dNTP’s?
On the 3′ end of a growing DNA chain
What is the only reaction catalyzed by DNA polymerase?
Nucleophilic attack of the 3’OH on the α-phosphate
of the incoming dNTP
What is DNA synthesized by?
DNA polymerase
What is incorporated into DNA during DNA synthesis?
deoxyribonucleoside triphosphates (dNTPs)
Is DNA synthesis endergonic or exergonic/ and why?
exergonic, because the high energy phosphate bonds are hydrolyzed in the reaction
What was Matthew Meselson and Frank Stahl’s experiment designed to determine?
The mode of DNA synthesis
What was the protocol for Meselson/Stahl’s experiment?
The grew E. coli in the presence of “heavy” nitrogen (15N) to label the bacteria’s DNA.
-then moved the bacteria to a normal 14N-containing medium
-Analyzed DNA by density at different times after shift
to 14N
What were the results of the Meselson/Stahl experiment?
Consistent with the hypothesis for semiconservative DNA synthesis
How is the incoming nucleotide incorporated?
According to base pairing rules
The Hershey-Chase experiment answered the question of whether protein or DNA was the genetic material by learning whether ______.
DNA or protein from a virus entered bacterial cells during infection
Okazaki fragments are found ______.
only on the lagging strand
Telomerase is needed to ______.
prevent the loss of DNA bases at the ends of linear chromosomes
What would happen if there wasn’t any telomerase?
linear chromosomes would shorten after each replication and eventually disappear
What aspect of DNA structure makes it possible for the enzymes of nucleotide excision repair to recognize many different types of DNA damage?
the regularity of DNA’s overall structure
Assuming that each replication fork moves at a rate of 500 base pairs per second, how long would it take to replicate the E. coli chromosome (with 4.6 million base pairs) from a single origin of replication?
4600 seconds

4,600,000/500=9200seconds
Each cycle does half, so divide by 2= 4600 seconds

What is the difference between DNA polymerase proofreading and excision repair?
Proof reading happens during DNA synthesis
Proof reading generally replaces a single mismatched base
Excision repair may excise a segment of damaged DNa
What does the nucleotide excision
repair system do?

recognizes DNA damage such as thymine dimers
What removes single-stranded DNA in a damaged section?
Enzymes
What are two things that can alter or damage DNA?
Chemicals or radiation
What does UV light do?
It causes thymine dimers to form, and the dimers then produce a kink in the DNA strand
When does the replication bubble form?
At the origin of replication
Which direction does the replication bubble grow in?
Both directions
How does DNA have to unwind?
locally during DNA replication to separate DNA strands
What is bidirectional replication?
When DNA replication occurs in both directions
Where does the replication process start in bacterial chromosomes?
At a single origin
What do proteins do for DNA during replication?
They are responsible for opening and stabilizing the reactions
What does the enzyme helicase do?
catalyzes the breaking of hydrogen bonds to separate the two DNA strands
What keeps the strands separated?
Single-strand DNA-binding proteins (SSBPs)
What does unwinding the DNA helix create?
Tension further down the helix
What cuts and rejoins the DNA?
topoisomerase
Where does topoisomerase work and why?
Further down on the replication fork, and it relieves tension in the helix
Where does DNA synthesis occur?
At replication forks
Replication forks:
A Y-shaped region where the DNA helix is separated into two strands on either side of the replication bubble
What is a problem with the replication fork?
One strand is synthesized in the direction of movement of the fork, while the other is not
Leading Strand:
Strands synthesized in the direction of the fork
Lagging Strand:
Strand synthesize in the opposite direction of the fork
In what manner is a leading strand synthesized?
A continuous manner
In what manner is the lagging strand synthesized?
A discontinuous manner
Okazaki fragments:
Short pieces of DNA that have to be initiated over and over again
What does DNA polymerase require to initiate DNA synthesis?
A primer
What is a primer?
a small oligonucleotide bonded to a template strand
Oligonucleotide:
a polynucleotide whose molecules contain a relatively small number of nucleotides.
What does a primer provide?
a free 3ʹ hydroxyl (OH) group that form a
phosphodiester bond with an incoming dNTP
What does primase do?
Synthesizes a short RNA segment that serves as a
primer
What does DNA polymerase III add?
bases to the 3′ end of the primer
How does synthesizing of the lagging strand start?
– Primase synthesizes a short RNA, a primer
What is the second step in synthesizing the lagging strand?
– DNA polymerase III adds bases to the 3ʹ end of the
primer forming an Okazaki fragment
What does DNA polymerase I remove?
the RNA primer at the
beginning of each Okazaki fragment and fills the gap
What does DNA ligase join?
the Okazaki fragments to form a
continuous DNA strand
Replisome:
A large, multi-enzyme machine responsible for DNA
synthesis at the replication fork
Why is the lagging strand synthesized in Okazaki fragments?
Because DNA polymerase has to start over and over to keep up with the replication fork
Telomeres:
the regions at the ends of linear
chromosomes
What do telomeres consist of?
short, repeating stretches of bases
Do telomeres contain genes?
No
What happens when the replication fork reaches the end of a linear chromosome?
Can’t prime on the end of the lagging DNA strand
What does telomerase add?
more repeating bases to the end of the lagging
strand
What does telomerase catalyze?
the synthesis of DNA from an RNA template carried with it
What makes the RNA primer?
Primase
What enzyme checks for mismatches pairs in the DNA?
polymerase
What happens in polymerase finds a mismatch?
It pauses and removes the mismatched base that
was just added
What is the average error rate of DNA replication?
less than one mistake per billion bases
What problem is encountered in replicating DNA at the ends of chromosomes (telomeres)?
Run out of template to prime DNA synthesis on the lagging strand

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