Chapter 10

Question 1

How many sites of replication are found in eukaryotes? Prokaryotes?

Answer 1

In eukaryotes, there can be multiple sites of replication (replicons)

In prokaryotes, there is usually only 1 site of replication (replicon)
Ex. OriC in e. coli

Question 2

What are the 2 conserved repeat sequences found in OriC in e.coli?

Answer 2

13-bp sequence
9-bp sequence

Question 3

Explain the 13-bp repeat sequence found in e.coli’s OriC.

Answer 3

One 13-bp sequence is present as three tandem repeats.

These three repeats are rich in A:T base pairs, facilitating the formation of a localized region of strand separation referred to as the replication bubble.

(A:T base pairs are held together by only two hydrogen bonds; come apart more easily.)

Question 4

Explain the 9-bp repeat sequence found in e.coli’s OriC.

Answer 4

The 9-bp sequence that is repeated four times.

These four sequences are binding sites for a protein that plays a key role in the formation of the replication bubble.

Question 5

What is topoisomerase and what does it do?

Answer 5

Topoisomerase is an enzyme which is responsible for the swivel that causes the transient single-strand break (cleavage of one phosphodiester bond in one strand of the double helix).

This causes the parental double helix to rotate 360° to unwind each gyre of the helix.

Question 6

What is a replicon?

Answer 6

A unit of DNA which is controlled by the origin of replication
ex.
Eukaryotes have 1
Prokaryotes have multiple

Question 7

What does ARS stand for?

Where do they occur?

What are they?

What are they made up of?

Answer 7

1. Autonomously Replicating Sequences (ARS)
2. They occur is yeast cells
3. They are segments of chromosomal DNA that allow a fragment of circularized DNA to replicate as an independent unit.(extrachromosomal self-replicating units)
4. They are 50 base pairs long and include a core 11-bp AT-rich sequence that contains either purines (Pu) or pyrimadines (Py)
   ex. ATTTATPuTTTA
   TAAATAPyAAAT

Question 8

How was the gross structure of a replicating bacterial chromosome discovered?

What did it show?

Answer 8

How?
E. coli cells were grown in a medium containing 3 H-thymidine for varying periods of time and were lysed gently so as not to break the chromosomes. They then carefully collected the chromosomes on membrane filters that were affixed to glass slides, coated with emulsion sensitive to β-particles and stored in the dark to allow sufficient radioactive decay.

What it showed?
The autoradiographs showed that the chromosomes of E. coli are circular structures that exist as θ-shaped intermediates during
replication. Also, the unwinding of the two complementary parental strands and their semiconservative replication occur simultaneously or are closely coupled.

Question 9

What is bacteriophage lambda (phage λ)?

What is it made up of?

What happens when it is injected into its host cell?

Answer 9

1. A small virus that infects E. coli
2. It contains a single linear molecule of DNA only 17.5 μm long.
   It has a single-stranded region, 12 nt long, at the 5′ end of each complementary strand called “sticky” ends.
   The ends can base-pair to form a hydrogen-bonded circular structure.
3. It forms a covalently closed circular molecule. The conversion from the hydrogen-bonded circular form to the covalently closed circular form is catalyzed by DNA ligase.
   Like the E. coli chromosome, it replicates in its circular form via θ-shaped intermediates.

Question 10

How do the enzymes that catalyze DNA synthesis add nucleotides?

Answer 10

DNA polymerases can only add nucleotides onto the 3′ end of a DNA strand—that is, they
synthesize DNA only in the 5′ - 3′ direction

Question 11

In prokaryotes, at each replication fork, the two progeny strands are extended in different ways.

What are they?

What are their differences?

Answer 11

The leading strand:
-It is extended continuously by the addition of nucleotides to its 3′ end.
-Moves (5’-3’)
-Synthesis activity is moving toward the fork

The lagging strand:
-It is extended discontinuously by DNA synthesis in spurts.
-Moves (3’-5’)
-Synthesis is moving away from the fork
-It grows by the synthesis of short segments of DNA, each of which is extended by the addition of nucleotides to its 3′ end; then the many segments are joined into one long, continuous chain.

Question 12

What links the Okazaki fragments together to produce the large DNA strands present in mature chromosomes?

What does it do?

How does it do it?

What can it not do?
(prokaryotes)

Answer 12

1. DNA Ligase
2. It catalyzes the covalent closure of nicks (missing phosphodiester linkages) in DNA molecules by using energy from NAD or ATP.
3. The Adenosine monophosphate (AMP) of the ligase-AMP intermediate forms a phosphoester linkage with the 5′-phosphate at the nick. Then a nucleophilic attack by the 3′-OH at the nick on the DNA-proximal phosphorus atom produces a phosphodiester
   linkage between the adjacent nucleotides at the site of the nick.
4. DNA ligase cannot fix breaks in DNA where one or more nucleotides are missing (gaps). Gaps can be filled in and sealed only by DNA polymerase AND DNA ligase.

Question 14

In the replication of E. coli, the replication bubble is formed by the interaction of prepriming proteins with oriC.

What are the steps involved in prepriming?

What is the importance of the DnaA protein?
(prokaryotes)

Answer 14

1. The binding of four molecules of DnaA protein to the four 9-base-pair (bp) repeats in oriC.
2. DnaA proteins bind cooperatively (one adds; more add) to form a core of 20 to 40 polypeptides with oriC DNA wound on the surface of the protein complex.
3. Strand separation begins within the three tandem 13-bp repeats in oriC and spreads until the replication bubble is created.
4. A complex of DnaB protein (DNA helicase) and DnaC protein (six molecules) joins the initiation complex and contributes to the formation of two bidirectional replication forks.

The DnaA protein appears to be largely responsible for the localized strand separation at oriC during the initiation process.

Question 15

What is an absolute requirement for DNA polymerase to function?

Answer 15

All DNA polymerases have an absolute requirement for a free 3′-OH on the end of the DNA strand being extended and an appropriate DNA template strand for activity.

No known DNA polymerase can initiate the synthesis of a new strand of DNA without a 3′-end to work on.

Question 16

Since no known DNA polymerase can initiate the synthesis of a new strand of DNA without a 3′-end to work on, some special mechanism must exist to initiate or prime the synthesis of new DNA chains once a replication bubble has formed.

What is this mechanism?

What do the primers do?

What is the primer in prokaryotes?

Answer 16

1. Each new DNA chain is initiated by
   a short RNA primer synthesized by DNA primase.
   2.The RNA primers provide the free 3′-OHs required for covalent extension of polynucleotide chains by DNA polymerases.
2. DNA polymerase III

Question 17

What primer is used in Prokaryotes?

What does it do?

What causes it to stop?

What occurs when it is stopped?

What takes over once the primers are replaced?

Answer 17

1. DNA polymerase III
2. It catalyzes the addition of deoxyribonucleotides to RNA primers, either continuously on the leading strand or discontinuously by the synthesis of Okazaki fragments on the lagging strand.
3. DNA polymerase III stops extending an Okazaki fragment when it bumps into the RNA primer of the preceding Okazaki fragment.

4.The RNA primers are excised and replaced with DNA chains.

5.DNA polymerase I

Question 18

What activities does DNA Polymerase I have in prokaryotes?

Answer 18

It contains three distinct enzyme activities :
1. 5′ - 3′ polymerase activity (replaces RNA with a DNA chain by using the adjacent Okazaki fragment with its free 3′-OH
as a primer)

2. 5′ - 3′ exonuclease activity (excises the RNA primer)
3. 3′ - 5′ exonuclease activity (cleaves off nucleotides from the 3′ termini of DNA strands)

Question 19

What causes the unwinding of DNA?

How does it unwind the DNA?
(prokaryotes)

Answer 19

1. Enzyme DNA helices (product of the dnaB gene)
2. It unwinds DNA molecules using energy from ATP.

Question 20

Once the DNA strands are unwound, they must be kept in an extended single-stranded form for replication.

How is this accomplished?
(prokaryotes)

Answer 20

They are maintained in this state by a
coating of single-strand DNA-binding protein (SSB protein)

Question 21

Recall that the E. coli chromosome contains a circular molecule of DNA. What provides the swivel or axis of rotation that prevents the DNA from becoming tangled (positively supercoiled) ahead of the replication fork?

How do these enzymes work?

Answer 21

1. The required axes of rotation during the replication of circular DNA molecules are provided by enzymes called DNA topoisomerases.
2. They catalyze transient breaks in DNA molecules but use covalent linkages to themselves to hold on to the cleaved molecules.

a. DNA topoisomerase I
b. DNA topoisomerase II

Question 22

Simply put, the formation of functional
template DNA requires what?

Answer 22

(a) DNA helicase, which unwinds the parental double helix

(b) Singlestrand DNA-binding (SSB) protein, which keeps the unwound DNA strands in an extended form.

Question 23

Explain the difference between DNA topoisomerase I and DNA topoisomerase II.

Answer 23

DNA topoisomerase I:
-Induce transient single-strand breaks

-Provides an axis of rotation that allows the segments of DNA on opposite sides of the
break to spin independently, with the phosphodiester bond in the intact strand serving as a swivel

-Energy-efficient because they conserve the energy of the cleaved phosphodiester linkages by storing it in covalent linkages between themselves and the phosphate groups at the cleavage sites; they then reuse this energy to reseal the breaks.

DNA topoisomerase II:
-Induce transient double-strand breaks

-They add negative supercoils or remove positive supercoils two at a time by an energy
(ATP)-requiring mechanism

-They carry out this process by cutting both strands of DNA, holding on to the ends at the cleavage site via covalent bonds, passing the intact double helix through the cut, and resealing the break

-Can separate interlocking circular molecules of DNA

Question 24

What is the type II topoisomerase enzyme used in E. coli? (prokaryote)

What does it do?

How does it function to prevent improper unwinding?

Answer 24

1. DNA Gyrase
2. The negative supercoils in bacterial chromosomes are introduced by DNA gyrase,
   with energy supplied by ATP.
3. Instead of creating positive supercoils ahead of the replication fork by unwinding the complementary strands of relaxed DNA, replication may produce relaxed DNA ahead of the fork by unwinding negatively supercoiled DNA.

Question 24

All polymerases require preexisting DNA with two essential components.

What are they?

What do they do?

Answer 24

1. A primer function:
   The primer DNA provides a terminus with a free 3′-OH to which nucleotides are added during DNA synthesis. They catalyze the formation of a phosphodiester bridge between the 3′-OH at the end of the primer DNA chain and the 5′-phosphate of the incoming deoxyribonucleotide.
2. A template function:
   The template DNA provides the nucleotide sequence that specifies the complementary sequence of the growing DNA chain.

Question 25

What activities does DNA polymerase III have?

Answer 25

1. 5′ - 3′ polymerase activity
2. 3′ - 5′ exonuclease activities
3. 5′ - 3′ exonuclease that is active only on single-stranded DNA

Question 26

How many DNA polymerases does e. coli have?
What are their functions?

Answer 26

1. 5 different types:
   DNA pol I: DNA repair enzymes

DNA pol II: DNA repair enzymes and replication of damaged DNA

DNA pol III: a complex enzyme composed of many different subunits

DNA pol IV: replication of damaged DNA

DNA pol V: replication of damaged DNA

Question 27

How many DNA polymerases are found in eukaryotes?

What are some of their functions?

Answer 27

Around 15:
-DNA polymerases (α, δ, and/or ε) work together to carry out the semiconservative
replication of nuclear DNA
-DNA polymerase γ (gamma) is responsible for the replication of DNA in mitochondria

Question 28

DNA polymerase III, the “replicase” in E. coli, is a multimeric enzyme in its complete or holoenzyme form.

What makes up the catalytic core?

Why is it not the best option?

What makes up the holoenzyme?

Answer 28

1. The catalytic core:
   -Contains three subunits:
   α (the dnaE gene product)
   ε (the dnaQ product)
   θ (the holE product)
   Addition of the τ subunit (the dnaX product) results in dimerization of the catalytic core and increased activity

2.The catalytic core synthesizes rather short DNA strands because of its tendency to fall off the DNA template.

3. The holoenzyme: (complete form)
   α, ε, θ, β, δ, γ
   The β-dimer forms a ring that encircles the replicating DNA molecule and allows DNA polymerase III to slide along the DNA while remaining tethered to it.

Question 29

Living organisms have solved the potential problem of insufficient fidelity during DNA replication by evolving a mechanism for proofreading the nascent DNA chain as it is being synthesized.

The proofreading process involves what?

How is the process carried out?

Answer 29

1. Scanning the termini of nascent DNA chains for errors and correcting them

2.This process is carried out by the 3′ - 5′ exonuclease activities of DNA polymerases

Question 30

What happens when a template-primer DNA has a terminal mismatch (an unpaired or
incorrectly paired base or sequence of bases at the 3′ end of the primer)?

What happens when it is fixed?

Answer 30

The 3′ - 5′ exonuclease activity of the DNA polymerase clips off the unpaired base or bases

When an appropriately base-paired terminus is produced, the 5′ 3′ polymerase activity of the enzyme begins resynthesis by adding nucleotides to the 3′ end of the primer strand

Question 31

What is the difference in enzymatic activity in monomeric enzymes vs. multimeric enzymes? (prokaryotes)

Answer 31

In monomeric enzymes like DNA polymerase I of E. coli, the 3′ 5′ exonuclease activity is built in.

In multimeric enzymes, the 3′ - 5′ proofreading exonuclease activity is often present on a separate subunit. In the case of DNA polymerase III of E. coli, this proofreading function is carried out by the ε subunit.

Question 32

Which polymerases in eukaryotes contain proofreading exonuclease activity?

Answer 32

DNA polymerases γ, δ, and ε contain 3′ 5′ proofreading exonuclease activities

Polymerases α and β lack this activity

Question 33

What starts the initiation of Okazaki fragments on the lagging strand? (prokaryotes)
What makes up this complex?
How does it work?

Answer 33

1. It is carried out by the primosome
2. A protein complex containing DNA primase and DNA helicase

3a. The primosome moves along a DNA molecule, powered by ATP.

b. As it proceeds, DNA helicase unwinds the parental double helix, and DNA primase synthesizes the RNA primers.

c. The RNA primers are covalently extended with the addition of deoxyribonucleotides by DNA polymerase III.

d. DNA topoisomerases provide transient breaks in the DNA that serve as swivels for DNA unwinding and keep the DNA untangled.
e. Single-strand DNA binding protein coats the unwound DNA and keeps it in an extended state for DNA polymerase III.

f. The RNA primers are replaced with DNA by DNA polymerase I.

g. The single-strand nicks left by polymerase I are sealed by DNA ligase

Question 34

What is the name of the complete apparatus that moves along the replication fork? (prokaryotes)

What does it contain?

What formation does it take?

Answer 34

1. The replisome
2. The replisome contains:
   -The DNA polymerase III holoenzyme
   -Two catalytic cores (one replicates the leading strand, the second the lagging strand)
   -The primosome
3. For the two catalytic cores of the polymerase III holoenzyme to synthesize the nascent leading and lagging strands, the lagging strand is thought to form a loop from the primosome to the second catalytic core of DNA polymerase III

(refer to the photo of the e.coli replisome)

Question 35

In e.coli, where does the termination of replication occur?
How do these sites work?
What other enzyme accompany them?

Answer 35

1. At variable sites within regions called terA and terB
2. They block the movement of replication forks advancing in the counterclockwise and clockwise directions, respectively.
3. DNA topoisomerases or special recombination enzymes then facilitate the separation of the nascent DNA molecules.

Question 36

What is rolling-circle replication used for?
What is it used for?
What is the main overview?
How does it work?
What is the outcome of the replication?
How are either achieved?

Answer 36

1. A mechanism for replicating circular DNA
2. It is used by:
   -Viruses to duplicate their genomes

-Bacteria to transfer DNA from donor cells to recipient cells during genetic exchange

-Amphibians to amplify extrachromosomal DNAs carrying clusters of ribosomal RNA genes during oogenesis

3. One parental circular DNA strand remains intact and rolls while serving as a template for the synthesis of a new complementary strand.
4. Replication is initiated when a sequence-specific endonuclease cleaves one strand at the origin, producing 3′-OH and 5′-phosphate termini. The 5′ terminus is displaced from the circle as the intact template strand turns about its axis. Covalent extension occurs at the 3′-OH of the cleaved strand.
5. It can produce either single-stranded or double-stranded progeny DNAs.
6. Single-stranded progeny molecules are produced by site-specific cleavage of the single-stranded tails at the origins of replication and recircularization of the resulting unit-length molecules.

Double-stranded progeny molecules, the singlestranded tails are used as templates for the discontinuous synthesis of complementary strands prior to cleavage and circularization.

Question 38

How does DNA replication work in the cell cycle with eukaryotes?

What are the stages of the cell cycle?

What are the check points?

Answer 38

1. DNA replication is restricted to the S phase of the cell cycle (synthesis)
2. G1 phase (immediately following the completion of mitosis)
   S phase
   G2 phase (preparation for mitosis)
   M phase (mitosis)
3. Entry into S phase and entry into mitosis. These checkpoints help to ensure that the DNA replicates once and only once during each cell division

Question 39

The replication of chromosomal DNA
in eukaryotes requires the activity of three different DNA polymerases.
What are they?
What are their functions?
What is PCNA?
What is Rf-C used for?

Answer 39

1. Polymerase α (Pol α)
   Polymerase δ (Pol δ)
   Polymerase ε (Pol ε)

2a. Pol α:
-The initiation of replication at origins

-The priming of Okazaki fragments during the discontinuous synthesis of the lagging strand.

-Exists in a stable complex with DNA primase; (The primase synthesizes the RNA primers, which are extended with deoxyribo nt. by Pol α to produce an RNA–DNA chain)

b. Pol δ:
-Completes the replication of the lagging strand

-Must interact with proteins PCNA and Rf-C to be active.

c. Pol ε:
-Catalyzes the replication of the leading strand

3. PCNA: (proliferating cell nuclear antigen)
   -A sliding clamp that tethers Pol δ to the DNA to allow processive replication (prevents DNA pol from falling off).

PCNA is a trimeric protein that forms a closed ring.

4. Rf-C: (replication factor C)
   -Required for PCNA to load onto the DNA.

-Induces a change in the conformation of PCNA that allows it to encircle DNA, providing the essential sliding clamp

Question 40

Why do the DNA polymerases in eukaryotes require different nucleases to remove RNA primers?

What nucleases are used instead?

Answer 40

1. Pol δ and ε do not have 5′ - 3′ exonuclease activity; thus, they cannot remove RNA primers like DNA polymerase I of E. coli.
2. RNA primers are excised by two nucleases:
   -Ribonuclease H1 (degrades RNA present in RNA–DNA duplexes)

-Ribonuclease FEN-1 (F1 nuclease 1).

Question 41

Nucleosomes must be disassembled to let the replisome duplicate the DNA packaged in them and then be quickly reassembled. A number of proteins are involved in the disassembly and assembly of nucleosomes during chromosome replication in eukaryotes.

What are the two most important proteins?

What do they do?

Answer 41

1. Nap-1 (nucleosome assembly protein-1)
   CAF-1 (chromatin assembly factor-1)

2a. Nap-1:
-transports histones from their site of synthesis in the cytoplasm to the nucleus

b. CAF-1:
-Carries them to the chromosomal sites of nucleosome assembly

-Delivers histones to the sites of DNA replication by binding to PCNA

Question 42

What is Telomerase?

What is the tandem repeat found in telomeres of humans?

How does telomerase work?

Answer 42

1. An RNA-containing enzyme called
   telomerase which forestalls the shortening of chromosome ends by protecting them from
   degradation.

2.TTAGGG

3. It recognizes this G-rich telomere sequence on the 3′ overhang and extends it 5′ - 3′ one repeat unit at a time.

-It does not fill in the gap opposite the 3′ end of the template strand; it extends the 3′ end of the template strand.

-It contains a built-in RNA template.