Chapter 12: DNA Replication and Recombination

Why must DNA replication be extremely accurate?

Because even a small error rate would create thousands of mistakes each time a cell divides, which would be catastrophic.

What example in the notes shows how damaging replication errors could be?

An error rate of one mistake per million base pairs would produce about 6,400 mistakes every time a cell divides.

Why must DNA replication also occur at high speed?

Because large genomes must be copied quickly enough for cell division to proceed efficiently.

What example is used to show the speed of DNA replication in E. coli?

E. coli has about 4.6 million base pairs and replicates at roughly 1,000 nucleotides per second.

What are the three proposed models of DNA replication mentioned in the notes?

Conservative, dispersive, and semiconservative replication

What is conservative replication?

It is the model in which the original double-stranded DNA molecule stays fully intact and serves as a template for a completely new double-stranded molecule.

What would happen to the original DNA molecule in conservative replication?

It would remain fully conserved through replication.

In conservative replication, what happens over additional rounds of replication?

The proportion of molecules containing new DNA increases, but the number of molecules with the original DNA remains constant.

What is dispersive replication?

It is the model in which both original strands break into fragments, those fragments serve as templates for new DNA, and the pieces reassemble into complete molecules.

What would each DNA molecule contain in dispersive replication?

Each resulting DNA molecule would contain interspersed fragments of old and new DNA.

What happens to the proportion of new DNA in dispersive replication after each round?

The proportion of new DNA within each molecule increases with each replication.

What is semiconservative replication?

It is the model in which the two original DNA strands separate, and each serves as a template for synthesis of a new complementary strand.

What happens after additional rounds of semiconservative replication?

More and more molecules containing entirely new DNA are produced.

Which model of DNA replication is correct?

DNA replication occurs in a semiconservative manner.

What was the purpose of the Meselson and Stahl experiment?

It was designed to determine which of the three models of DNA replication applied to E. coli cells.

What two nitrogen isotopes were used in the Meselson and Stahl experiment?

Heavy nitrogen, ¹⁵N, and light nitrogen, ¹⁴N

Why were ¹⁵N and ¹⁴N useful in the Meselson and Stahl experiment?

They allowed researchers to distinguish old DNA from newly synthesized DNA based on density.

How was the original E. coli culture prepared in the Meselson and Stahl experiment?

It was grown for many generations in a medium containing ¹⁵N as the sole nitrogen source.

What happened to the bacteria after growth in ¹⁵N medium?

All cells incorporated ¹⁵N into their purines and pyrimidines.

What change was made after the bacteria had incorporated ¹⁵N?

The remaining cells were transferred to a medium containing only ¹⁴N.

In the Meselson and Stahl experiment, what kind of DNA was considered heavy?

DNA synthesized before the switch, containing ¹⁵N, was heavy.

In the Meselson and Stahl experiment, what kind of DNA was considered light?

DNA synthesized after the switch, containing ¹⁴N, was light.

What method was used to separate heavy and light DNA in the Meselson and Stahl experiment?

Equilibrium density gradient centrifugation.

How does equilibrium density gradient centrifugation separate DNA?

DNA fragments move until they reach the position where their density matches the surrounding solution; light molecules rise higher and heavy molecules sink lower.

What result was seen after one round of replication in the Meselson and Stahl experiment?

A single intermediate band appeared between the heavy and light positions.

Why did the intermediate band support semiconservative replication?

Because it showed that each DNA molecule contained one old heavy strand and one new light strand.

What conclusion was made from the Meselson and Stahl experiment?

E. coli replicates its DNA semiconservatively.

Why did the Meselson and Stahl result rule out conservative replication after one generation?

Because conservative replication would have produced separate heavy and light bands, not a single intermediate band. This is also illustrated by the diagram on page 1.

Why did the Meselson and Stahl result rule out dispersive replication over successive generations?

Because semiconservative replication predicts increasing numbers of fully light molecules over time, unlike the persistent mixed pattern expected from dispersive replication.

What is a replicon?

A replicon is a segment of DNA that undergoes replication and contains an origin of replication.

What does replication within a replicon do?

It starts at the origin and continues until the entire replicon has been copied.

What does the notes mean by “modes of replication”?

They mean the different ways semiconservative replication can occur in circular and linear DNA molecules.

What is theta replication?

Theta replication is a common mode of replication in circular DNA.

Why is theta replication called “theta” replication?

Because the intermediate structure formed during replication resembles the Greek letter theta, as shown in the page 2 diagram.

What happens at the origin during theta replication?

Double-stranded DNA unwinds at the origin, producing strands that can serve as templates.

What is a replication bubble?

It is the loop formed when DNA unwinds during replication.

What is a replication fork?

It is the point where the two DNA strands separate from the helix during replication.

What is unidirectional theta replication?

It is theta replication in which only one replication fork is present and moves around the circle in one direction.

What is bidirectional theta replication?

It is theta replication in which two forks form at opposite ends of the replication bubble and move outward in both directions until they meet.

What are the products of theta replication?

Two circular DNA molecules

What type of DNA commonly undergoes rolling-circle replication?

It occurs in some viruses and in the F factor of E. coli.

How is rolling-circle replication initiated?

It begins when one nucleotide strand is broken, exposing a 3′-OH group and a 5′-phosphate group.

During rolling-circle replication, where are new nucleotides added?

They are added to the 3′ end of the broken strand.

What serves as the template in rolling-circle replication?

The inner, unbroken strand serves as the template.

What happens to the 5′ end of the broken strand during rolling-circle replication?

It is displaced away from the template strand.

Why is it called rolling-circle replication?

Because the replication fork can continue around the circular template repeatedly, displacing the broken strand as synthesis proceeds

What can happen if the rolling-circle replication fork goes around the circle several times?

It can produce several linked copies of DNA.

What happens to the linked copies produced by rolling-circle replication?

They are eventually displaced and cleaved at the ends of the revolutions.

What are the products of rolling-circle replication?

One circular DNA molecule and one linear molecule that may later circularize

Why can’t large linear eukaryotic chromosomes be replicated quickly from just one origin?

Because they contain too much DNA to be copied fast enough from a single starting point.

How do eukaryotic cells solve the problem of replicating large linear chromosomes?

They initiate replication at thousands of origins.

What is the typical size range of eukaryotic replicons in the notes?

About 20,000 to 300,000 base pairs.

What forms at each origin during linear eukaryotic replication?

The DNA unwinds and produces a replication bubble.

How does replication proceed from each bubble in linear eukaryotic replication?

Replication occurs on both strands at each end of the bubble as two forks move outward.

What happens when neighboring eukaryotic replicons expand?

The replication forks of adjacent replicons eventually meet, and the replicons fuse.

What are the final products of linear eukaryotic replication?

Two identical linear DNA molecules, as shown by the page 2 figure and table.

What are the three major requirements for DNA replication?

A single-stranded DNA template, raw materials in the form of nucleotides, and enzymes or proteins that read the template and assemble the new strand.

Why must the template be single-stranded?

Because the bases must be exposed so that complementary nucleotides can be added.

What raw materials are used in DNA replication?

Deoxyribonucleoside triphosphates, or dNTPs.

Why doesn’t DNA synthesis happen spontaneously?

Because it requires enzymes and proteins to read the template and catalyze addition of nucleotides to the 3′-OH end.

n what direction does DNA polymerase add nucleotides to a growing strand?

It adds nucleotides only to the 3′ end of the growing strand.

In what overall direction is new DNA synthesized?

New DNA is synthesized in the 5′ to 3′ direction.

What is continuous replication?

It is replication in which the new strand is synthesized continuously as the DNA unwinds.

What is the leading strand?

It is the strand synthesized continuously because its template is oriented in the proper direction for ongoing synthesis.

What is discontinuous replication?

It is replication in which DNA is synthesized in short bursts because synthesis proceeds opposite to the direction of unwinding on that template.

Why can’t the lagging strand be synthesized continuously?

Because DNA polymerase can only synthesize 5′ to 3′, so it must repeatedly wait for more template to be exposed and then restart near the replication fork.

What is the lagging strand?

It is the strand synthesized discontinuously in short segments during DNA replication.

What are Okazaki fragments?

They are the short lengths of DNA produced during discontinuous replication of the lagging strand.

What ultimately happens to Okazaki fragments?

They are linked together to form a continuous DNA strand.

What are the four stages of bacterial DNA replication?

Initiation, unwinding, elongation, and termination.

What happens during initiation of bacterial DNA replication?

Initiator proteins bind the origin of replication and cause a short stretch of DNA to unwind.

What is the initiator protein called in E. coli?

DnaA.

What is the bacterial origin of replication in E. coli called?

oriC.

What is the minimal sequence length required for oriC to function?

245 base pairs.

Why must DNA be unwound before synthesis begins?

Because DNA polymerase needs a single-stranded template.

What is the role of DNA helicase?

It breaks the hydrogen bonds between the bases of the two DNA strands to separate them.

Where does helicase function during replication?

At the replication fork.

Which template strand does helicase bind in the bacterial system described in the notes?

It binds the single-stranded lagging-strand template and moves in the 5′ to 3′ direction.

What do single-strand-binding proteins do?

They bind to exposed single-stranded DNA after helicase unwinds it.

Why are single-strand-binding proteins important?

They protect the nucleotide chains and prevent secondary structures such as hairpins from forming.

What is the role of DNA gyrase?

It reduces torsional strain ahead of the replication fork.

How does DNA gyrase reduce torsional strain?

It makes a double-strand break, passes another part of the helix through the break, and then reseals the DNA.

What happens if gyrase is inhibited?

DNA synthesis and bacterial growth stop.

What happens during elongation?

Single-stranded DNA is used as the template for synthesis of new DNA.

Why can’t DNA polymerases start synthesis completely on their own?

Because they require an existing nucleotide with a 3′-OH group to which they can add the next nucleotide.

What enzyme makes the primer needed to start DNA synthesis?

Primase

What kind of molecule is the primer made of?

RNA

Why is primase able to start primer synthesis without a preexisting 3′-OH group?

Because it is an RNA polymerase

How many primers are needed on the leading strand?

Only one primer is needed at the 5′ end of the new leading strand.

How many primers are needed on the lagging strand?

A new primer is needed at the beginning of each Okazaki fragment.

What is the relationship between primase and helicase?

Primase forms a complex with helicase at the replication fork and moves along the lagging-strand template.

What is DNA polymerase III?

It is the main workhorse of bacterial DNA replication and a large multiprotein complex.

What does DNA polymerase III do?

It synthesizes DNA by adding nucleotides to the 3′ end of a growing strand.

Why must two DNA polymerase III units be present at the replication fork?

Because one is needed for each strand being synthesized.

What are the two enzymatic activities of DNA polymerase III?

5′ to 3′ polymerase activity and 3′ to 5′ exonuclease activity.

What does the 5′ to 3′ polymerase activity of DNA polymerase III do?

It adds new nucleotides to the growing DNA strand.

What does the 3′ to 5′ exonuclease activity of DNA polymerase III do?

It removes incorrectly paired nucleotides, allowing proofreading.

What does high processivity mean for DNA polymerase III?

It can add many nucleotides without releasing the template, often until replication of that template region is complete.

What is the main special role of DNA polymerase I in replication?

It removes RNA nucleotides of the primer and replaces them with DNA nucleotides using its 5’ → 3’ exonuclease activity.

What enzymatic activities does DNA polymerase I have?

It has 5′ to 3′ polymerase activity, 3′ to 5′ exonuclease activity, and 5’ to 3’ exonuclease activity