3.1 HS DNA structure and function

What were the two strains of Streptococcus pneumoniae used in Frederick Griffith's experiment?

The virulent (S) strain (smooth colony) and the non-virulent (R) strain (rough colony).

What was the critical observation Griffith made when he mixed heat-killed S cells with living R cells and injected the mixture into mice?

The mice died, and living virulent (S) bacteria were recovered from the mice.

What did Griffith conclude was responsible for changing the non-virulent R bacteria into virulent S bacteria?

Some unknown "transforming substance" (or principle) was passed from the dead S cells to the live R cells.

In Griffith's experiment, which component of the heat-killed S cells was suggested to be the genetic material, even though its identity wasn't yet known?

The "transforming substance".

What was the main goal of the experiment conducted by Avery, McLeod, and McCarty?

To identify the chemical nature of Griffith's "transforming substance".

Which purified macromolecule from the heat-killed S cells was shown to be the transforming agent in the Avery-McLeod-McCarty experiment?

DNA.

The addition of which enzyme to the transforming substance prevented the transformation of R cells into S cells, thus concluding DNA was the genetic material?

Deoxyribonuclease (DNase).

Why did the addition of proteases or ribonuclease (RNase) not prevent the bacterial transformation?

Because the transforming substance was DNA, not protein or RNA, so these enzymes did not destroy it.

Hershey and Chase used which biological agents to study the location of the genetic material?

Bacteriophages (viruses that infect bacteria).

How did Hershey and Chase differentially label the protein capsule and the DNA of the bacteriophage?

They labeled protein with radioactive sulfur and DNA with radioactive phosphorus.

After the bacteriophages infected the bacteria, which radioactive label was primarily found inside the bacterial cells?

Radioactive phosphorus, which labeled the DNA.

After the bacteriophages infected the bacteria, which radioactive label was primarily found outside the bacterial cells (in the supernatant)?

Radioactive sulfur, which labeled the protein.

What was the final conclusion of the Hershey-Chase experiment regarding the genetic material?

DNA, not protein, is the genetic material responsible for directing the production of new viruses.

What is the overall shape/structure of the DNA molecule according to the Watson-Crick model?

A double helix (a two-stranded spiral).

Where are the nitrogenous bases located within the double helix structure?

They are paired on the inside (the core) of the helix.

Where is the sugar-phosphate backbone located within the double helix structure?

On the outside of the helix, forming the two continuous strands.

What term describes the arrangement of the two DNA strands running in opposite directions?

Antiparallel orientation.

What functional group is found at the 5' end of a DNA strand?

A phosphate (PO4) group

What functional group is found at the 3’ end of a DNA strand?

A hydroxyl (OH) group

If one DNA strand runs in the 5' to 3’ direction, what is the direction of its complementary strand?

It runs in the 3' to 5’ direction

What is the complementary base for Adenine (A) in DNA?

Thymine (T).

What is the complementary base for Guanine (G) in DNA?

Cytosine (C).

Which rule, stating that the amount of A equals T and the amount of G equals C, is accounted for by the complementary base-pairing in the DNA model?

Chargaff's rules.

What type of chemical bond holds the complementary nitrogenous bases (A-T and C-G) together?

Hydrogen bonds.

How many hydrogen bonds form between Adenine (A) and Thymine (T)?

Two (2) hydrogen bonds.

How many hydrogen bonds form between Guanine (G) and Cytosine (C)?

Three (3) hydrogen bonds.

What were the three hypothesized modes of DNA replication that the Meselson-Stahl experiment was designed to distinguish?

Conservative, Dispersive, and Semi-conservative.

What was the purpose of labeling the original parental DNA with the heavy nitrogen isotope 15N before replication began?

To create a dense/heavy parental DNA molecule that could be distinguished from newly synthesized light DNA via density gradient centrifugation.

What would the predicted result (band pattern) be after one round of replication in the light 14N medium if the Conservative model was correct?

Two bands: one at the heavy (15N/15N) density and one at the light (14N/14N) density.

What was the actual result observed after one round of replication in the light (14N) medium?

A single band of intermediate density (15N/14N hybrid DNA).

The observation of a single, intermediate density band after the first round of replication immediately disproved which proposed model?

The Conservative model.

Based on the 15N/14N hybrid band observed after one round, what is the composition of the DNA molecules created by Semi-conservative replication?

Each molecule consists of one original heavy (15N) strand and one newly synthesized light (14N) strand.

If the Dispersive model was correct, what would the predicted result be after one round of replication in 14N medium?

A single band of intermediate density, as every strand would contain a mixture of 15N and 14N segments.

What was the critical observation after two rounds of replication (in the light 14N medium) that distinguished semi-conservative from dispersive replication?

Two bands were observed: one intermediate (15N/14N) and one light (14N/14N), in a 1:1 ratio.

If the Dispersive model had been correct, what would the band pattern look like after two rounds of replication in 14N medium?

A single band that is slightly lighter than the first round's intermediate band.

The final results of the Meselson-Stahl experiment overwhelmingly confirmed which mode of DNA replication?

Semi-conservative replication.

What is the initial, unwound section of DNA where replication takes place, and how does the process proceed from the origin?

A replication fork, and the process proceeds bidirectionally (in both directions).

Which enzyme is responsible for "unzipping" or unwinding the DNA double helix at the replication fork?

Helicase

Why is an RNA primer necessary before DNA polymerase can begin synthesizing a new strand?

DNA Polymerase can only add nucleotides to an existing 3'-OH (hydroxyl) group, which the primer provides.

Which enzyme synthesizes the necessary short RNA segment to start the DNA polymerization process?

Primase

What is the rule regarding the direction in which DNA Polymerase can add new nucleotides?

It can only add nucleotides to the 3' end, meaning new strands are synthesized exclusively in the 5' to 3' direction.

Which enzyme is responsible for the bulk of DNA synthesis, adding new nucleotides complementary to the template strand?

DNA Polymerase

What is the primary role of DNA Ligase during the replication process?

To seal the sugar-phosphate backbone gaps between the short Okazaki fragments on the lagging strand.

Predict the outcome if Helicase were nonfunctional or missing.

The DNA duplex would not be unwound, and the replication fork would fail to form or advance, stopping replication entirely.

Predict the outcome if Primase were nonfunctional or missing.

No new DNA strands could be started, because DNA Polymerase would lack the required RNA primer and its 3'-OH group to begin polymerization.

Predict the outcome if DNA Ligase were nonfunctional or missing.

The short Okazaki fragments on the lagging strand would be synthesized but would not be covalently linked to form a continuous strand.

What is the fundamental property of DNA Polymerase that necessitates the difference between leading and lagging strand synthesis?

It can only add new nucleotides to the 3'-OH end of an existing strand, meaning synthesis can only occur in the 5' to 3' direction.

What structural feature of the DNA template strands at the replication fork imposes the second constraint on replication?

The two strands are antiparallel (one runs 3' to 5' and the other runs 5' to 3').

What two factors together determine that one new strand will be continuous and the other discontinuous?

The 5' to 3’ directionality of DNA Polymerase and the antiparallel nature of the template strands.

Which template strand directionality (relative to the synthesis) is used to create the Leading Strand?

The template strand running 3' to 5'

Describe the synthesis of the Leading Strand.

It is synthesized continuously in the 5' to 3' direction, moving toward the advancing replication fork.

Which template strand directionality (relative to the synthesis) is used to create the Lagging Strand?

The template strand running 5' to 3'

Why must the Lagging Strand be synthesized in short segments?

Because DNA Polymerase must synthesize the new strand in the 5' to 3' direction, it must move away from the replication fork, leading to discontinuous synthesis.

What are the short DNA segments synthesized on the lagging strand called?

Okazaki fragments.

Which enzyme is responsible for forming the final covalent bond to connect the Okazaki fragments into a single continuous strand?

DNA Ligase