26. What are the major differences between helicases, topoisomerases, and ligase

Question

26. What are the major differences between helicases, topoisomerases, and ligases used in DNA replication?

a) Helicases and topoisomerases unwind DNA, with helicases functioning to relieve supercoil stress ahead of the replication process; and ligases seal DNA that has been broken or synthesized as Okazaki fragments.

b) All three unwind DNA, but helicases do so before the replication machinery, ligases with the machinery, and topoisomerases ahead of it.

c) Helicases and topoisomerases unwind DNA, with topoisomerases functioning to relieve supercoil stress ahead of the replication fork; and ligases seal DNA that has been broken or synthesized as Okazaki fragments.

d) Helicases and topoisomerases bind double-stranded DNA, and ligases bind RNA-DNA hybrids.

e) None of the above.

27. Aspartate and glutamate are commonly found in the active sites of enzymes that act upon DNA because:

a) Their acidic side chains are excellent nucleophiles to mediate phosphodiester bond breaking and joining reactions.

b) They carry a positive charge at physiological pH and therefore have a high affinity for the negatively charged backbone of DNA.

c) Their tendency to donate protons enables interaction with proton acceptors in the major groove.

d) They are able to coordinate divalent metal cations, which in turn activate molecules that form nucleophiles.

e) They are highly acidic and therefore are able to break DNA bonds.

28. You are characterizing a new DNA polymerase. When the enzyme is incubated with [32P] DNA and no dNTPs, you observe the release of [32 P]dNMPs. This release is prevented by adding unlabeled dNTPs. Which of the following describes the reactions that most likely underlie these observations?

a) The DNA polymerase contains 3'-5' and a 5 1-3' exonucleases that degrade DNA to produce [32 P]dNMPs.

b) The DNA polymerase contains a 5'-3' exonuclease that degrades DNA to produce [32P]dNMPs. c) The DNA polymerase contains a 3'-5' exonuclease that degrades DNA to produce [32P]dNMPs.

d) Any of the above could explain these observations.

e) None of the above.

29. During assembly and growth of bacterial replication forks:

a) Four hexamers of DnaB helicase are assembled on the replication bubble, one on each

strand.

b) DnaB helicase helps load the leading strand POI Ill core onto the strand that will become the leading strand as the replication fork advanceé.

c) The coupled helicase-Pol Ill holoenzyme complex allows rapid synthesis of the leading strands, prior to initiation of lagging strand synthesis.

d) One ß clamp and one POI Ill core of the POI Ill holoenzyme load onto the leading strandprimer hybrid and initiate leading strand synthesis from each replication fork.

e) One ß clamp and one POI Ill core of the POI Ill holoenzyme load onto the leading strandprimer hybrid and initiate lagging strand synthesis from each replication fork.

30. Many of the proteins that have roles in DNA metabolism are AAA* ATPases. Which of the following descriptions is correct in regards to what is accomplished when ATP is hydrolyzed by each of the following AAA+ ATPases:

a) In DnaA, ATP hydrolysis activates the DnaA for replication initiation.

b) In DnaC, ATP hydrolysis helps release DnaB helicase as it is loaded onto the DNA.

c) In POI Ill y and subunits, ATP hydrolysis prevents the clamp from closing around the DNA.

d) In POI Ill polymerase core, ATP hydrolysis facilitates release of the polymerase from the ß clamp once the end of the Okazaki fragment is reached.

e) All of the above

Explanation / Answer

Answer for question number 26 : The correct option is B (All three unwind DNA, but helicases do so before the replication machinery, ligases with the machinery, and topoisomerases ahead of it.)

Supplementary Information :

Helicases are ATP dependent motor proteins that move directionally along a nucleic acid phosphodiester backbone and brings about the denaturation of DNA molecules using energy derived from ATP hydrolysis. The function of helicases is very important in processes such as DNA replication, transcription, recombination and DNA repair. In prokaryotes product of dnaB gene functions as helicase and in eukaryotes Mcm2-7 complex works as helicase.

Ligases catalyze the formation of phosphodiester bond between the 3’ OH and 5’ phosphate groups. DNA ligases are vital enzymes required for important cellular processes such as DNA replication, repair of damaged DNA and recombination. The enzyme mediates the formation of phosphodiester bonds between adjacent 3'-OH and 5'-phosphate termini, thereby joining the nicks in double stranded DNA. Ligases can be classified into two groups depending on their requirement for ATP or NAD+ as the cofactor. All eukaryotic and virally encoded enzymes are ATP-dependent, whereas most prokaryotic enzymes require NAD+ for their activity.

Topoisomerases are the enzymes that plays a important role in relieving the torsional strain generated because of the movement of helicase during denaturation of DNA for replication purposes. There are two classes of topoisomerases

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