Exam 3: 30 DNA Replication (2/3 complete)

Question 1

14. DNA replication initiation is highly regulated in both prokaryotes and eukaryotes. In prokaryotes, such as E. coli, only fully methylated origins are capable of initiation. Which enzyme is responsible for methylating all E. coli GATC sequences?

A) DnaA
B) Dam
C) MutL
D) MutS

Answer 1

B) Dam, The Dam enzyme is responsible for methylating GATC sequences in E. coli, a crucial regulatory mechanism for controlling DNA replication initiation.

DNA Adenine Methyltransferase (Dam): The sources explicitly state that Dam methylase is responsible for methylating all E. coli GATC sequences. This methylation occurs specifically at the adenine base within the GATC sequence.

Let’s examine why the other options are incorrect:
DnaA: DnaA is the initiator protein in E. coli and is responsible for binding to the origin of replication. While DnaA binding is essential for initiation, it does not directly methylate DNA sequences.

MutL and MutS: MutL and MutS are involved in DNA mismatch repair, a system that corrects errors that occur during DNA replication. [This information is not from the sources provided.] They do not play a role in DNA methylation.

Question 2

Which best describes the function of the Dam enzyme?

A) responsible for methylating all E. coli GATC sequences.

B) initiator protein in E. coli and is responsible for binding to the origin of replication.

Answer 2

A) responsible for methylating all E. coli GATC sequences.

Question 3

Which best describes the function of the DnaA protein?

A) responsible for methylating all E. coli GATC sequences.

B) initiator protein in E. coli and is responsible for binding to the origin of replication.

Answer 3

B) initiator protein in E. coli and is responsible for binding to the origin of replication.

Question 4

15. During DNA replication, there are two metal ions (Mg2+ or Mn2+) held in place by interaction with two highly conserved aspartate residues in the DNA polymerase. One metal ion primarily interacts with the 3’- OH (metal hydroxide), resulting a reduced association between the O and H. This produces a powerful nucleophile O-, performing nucleophilic attack.

Which phosphorus site is under such attach in the incoming nucleotide, leading to the formation of a phosphodiester bond?

A) 𝛼 phosphorus
B) β phosphorus
C) δ phosphorus
D) Either A or B

Answer 4

A) 𝛼 phosphorus

Metal ion A interacts with the 3’-OH group of the primer, the terminal nucleotide of the growing DNA strand.
This interaction leads to a reduction in the association between the oxygen and hydrogen of the 3’-OH, creating a more powerful nucleophile, the 3’-O-.
This activated 3’-O- performs a nucleophilic attack (SN2 reaction) on the α-phosphorus of the incoming deoxyribonucleoside triphosphate (dNTP).

Question 5

16. DNA topoisomerase II (a dimer) can untangle the two interlocked or crossed DNA double helices.

For each cycle (untangling), 2 ATPs are consumed. What is (are) the function(s) of ATP?

A) The energy of ATP hydrolysis is used to make a double strand (DNA) break on G segment

B) Drive protein conformational changes

C) Transfer pyrophosphate to T segment

D) All the above

Answer 5

B) Drive protein conformational changes

ATP Binding and Conformational Change: ATP binding to topoisomerase II is essential for its activity. The binding of ATP to the enzyme triggers a significant conformational change that is necessary for creating the double-strand break in the G segment. This conformational change is crucial for allowing the T segment to pass through the break.

Why the other options are wrong:
(A) The energy of ATP hydrolysis is used to make a double-strand (DNA) break on G segment: While ATP hydrolysis provides the energy for the overall process, it’s not directly used to break the DNA. The enzyme’s catalytic tyrosine residues perform the cleavage.

(C) Transfer pyrophosphate to T segment: There is no transfer of pyrophosphate from ATP to the T segment during the topoisomerase II reaction.

(D) All of the above: As explained above, only option (B) is correct.

Question 6

Clicker:
Helicase has a beautiful ring structure formed from six subunits. Each subunit uses a loop to bind the sugar phosphate backbone of DNA. Which of the following residues are likely to be found in these loops?

A. Ala and Leu
B. Ser and Asp
C. Lys and Arg
D. Tyr and Trp

Answer 6

C. Lys and Arg

Helicase Structure and Function: The sources describe DNA helicase as an essential enzyme in DNA replication. It unwinds the double-stranded DNA helix, separating the two strands to allow for replication. Helicase has a ring structure formed from six identical subunits. Each subunit contains a loop that binds to the sugar-phosphate backbone of the DNA.

Question 7

What does DNA helicase do?

A) seal together the Okazaki fragments
B) load sliding clamp to DNA
C) Open up the DNA helix

Answer 7

C) Open up the DNA helix

Question 8

What does the clamp loader do?

A) seal together the Okazaki fragments
B) load sliding clamp to DNA
C) Open up the DNA helix

Answer 8

B) load sliding clamp to DNA

Question 9

What does DNA ligase do?

A) seal together the Okazaki fragments
B) load sliding clamp to DNA
C) Open up the DNA helix

Answer 9

A) seal together the Okazaki fragments

Question 10

What does SSB do?

A) catalyze the elongation of the DNA strand

B) keep DNA polymerases firmly on the DNA

C) keep single-strand DNA in an extended, relatively inflexible conformation

Answer 10

C) keep single-strand DNA in an extended, relatively inflexible conformation

Question 11

What does DNA polymerase do?

A) catalyze the elongation of the DNA strand

B) keep DNA polymerases firmly on the DNA

C) keep single-strand DNA in an extended, relatively inflexible conformation

Answer 11

A) catalyze the elongation of the DNA strand

Question 12

What does sliding clamps do?

A) catalyze the elongation of the DNA strand

B) keep DNA polymerases firmly on the DNA

C) keep single-strand DNA in an extended, relatively inflexible conformation

Answer 12

B) keep DNA polymerases firmly on the DNA

Question 13

What does DNA primase do?

A) catalyze the formation of RNA primer
B) remove RNA primer

Answer 13

A) catalyze the formation of RNA primer

Question 14

What does RNAse H do?

A) catalyze the formation of RNA primer
B) remove RNA primer

Answer 14

B) remove RNA primer

Question 15

What is responsible for relieving helical winding and DNA tangling?

Answer 15

DNA topoisomerases

(II)

Question 16

DNA topoisomerase I is responsible for:

A) single strand break (solve winding problem)

B) double strand break (solve tangling problem)

Answer 16

A) single strand break (solve winding problem)

Question 17

DNA topoisomerase II is responsible for:

A) single strand break (solve winding problem)

B) double strand break (solve tangling problem)

Answer 17

B) double strand break (solve tangling problem)

Question 18

True or False:
All of the following require ATP.
-DNA helicases
-Clamp loader
-DNA topoisomerase II
-DNA ligase

Answer 18

True

Question 19

Clicker:
The chemotherapy medication etoposide acts by inhibiting

A) Topoisomerase I
B) Topoisomerase II
C) Helicase
D) DNA Ligase

Answer 19

B) Topoisomerase II

The sources clearly identify etoposide as an inhibitor of topoisomerase II. This inhibition leads to the accumulation of double-strand DNA breaks, making etoposide an effective chemotherapy drug for targeting rapidly dividing cancer cells.

Question 20

What are the substrates for DNA polymerase?

Answer 20

• Template strand
• Primer with a free -OH group at the 3’ end of a polynucleotide chain
  -Deoxyribosnucleoside triphosphates (dNTPs) to extend the DNA strand

Question 21

(Question from discussion)
26. Which of the following correctly describes the catalytic strategy used by DNA polymerase to speed up the reaction?

A) DNA polymerase forms a transient covalent bond with the incoming nucleotide

B) DNA polymerase possesses two metal ions, one activating the 3’-OH and one stabilizing the leaving group, i.e., pyrophosphate

C) DNA polymerase uses metal ion catalysis. Specifically, the metal ions form transient covalent bond with a heme group present in DNA

D) DNA polymerase only provides a meeting place that allows nucleotide to bind to
the primer strand

Answer 21

B) DNA polymerase possesses two metal ions, one activating the 3’-OH and one stabilizing the leaving group, i.e., pyrophosphate

• DNA polymerase uses two metal ions that are held in place by interactions with two aspartate residues.
• One metal ion interacts primarily with the 3’-OH, producing a powerful nucleophile O- to perform an SN2 attack to the 𝛼 phosphorus.
• The other metal ion interacts with the incoming dNTP to position it for nucleophilic attack and to stabilize the resulting –PP (pyrophosphate).

Let’s look at why the other options are incorrect.

A) DNA polymerase forms a transient covalent bond with the incoming nucleotide.** This statement is incorrect. DNA polymerase does not form a covalent bond with the incoming nucleotide. It facilitates the formation of a phosphodiester bond between the incoming nucleotide and the primer strand.

C) DNA polymerase uses metal ion catalysis. Specifically, the metal ions form a transient covalent bond with a heme group present in DNA.** This statement is incorrect. DNA does not contain a heme group. Heme groups are found in proteins like hemoglobin and myoglobin and are involved in oxygen binding. [outside source] Metal ions in DNA polymerase interact with the phosphate groups of the incoming dNTP and the 3’-OH of the primer strand.

D) DNA polymerase only provides a meeting place that allows nucleotide to bind to the primer strand.** This statement is incorrect. While DNA polymerase does provide a binding site for the template strand, primer strand, and incoming nucleotide, it also plays a crucial catalytic role in facilitating the nucleophilic attack and stabilizing the transition state during phosphodiester bond formation.