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GATE 2020 BT Q.37: solution and answer

GATE 2020 · BTQ.37 · 2 marks · MCQ

The schematic of a plasmid with a gap in one of the strands is shown below: [circular double-stranded plasmid diagram]. Top strand (continuous, outer): 5'-ATCGCGATG-3'. Bottom strand (broken, shown below it): left fragment 'TAGC' ending in a vertical line down to '3'OH', then a gap, then right fragment 'AC' with a vertical line down to '5'OH'. So the bottom strand is interrupted: one piece ends 3'-OH (after TAGC), and the next piece begins 5'-OH (before AC), with several bases of the complementary strand missing between them (a gap), opposite the continuous top strand ATCGCGATG. Which of the following enzyme(s) is/are required to fill the gap and generate a covalently closed circular plasmid? [P] DNA ligase [Q] Alkaline phosphatase [R] DNA polymerase [S] Polynucleotide kinase

GATE 2020 BT question 37: A circular plasmid drawn as two concentric circles (double-stranded DNA, most of the circle shown as plain double lines…Tap to open full size
Figure from the GATE 2020 BT paper. It is part of the question.
Figure description

A circular plasmid drawn as two concentric circles (double-stranded DNA, most of the circle shown as plain double lines representing the rest of the plasmid). At one point on the circle, the sequence is spelled out: the outer/top strand reads 5'-ATCGCGATG-3' continuously (no break). Directly below it, aligned by base-pairing, the bottom strand is shown broken into two fragments with a gap between them: the left fragment reads 'TAGC' and has a line dropping down to the label '3'OH' (marking its 3' terminus); after a gap of missing bases, the right fragment reads 'AC' and has a line dropping down to the label '5'OH' (marking its 5' terminus, explicitly written as 5'-OH rather than 5'-phosphate). This depicts a plasmid strand with both a gap (missing nucleotides) and, once the gap is filled, a nick whose 5' end lacks a phosphate group.

  1. (A)

    P only

  2. (B)

    P, R and S only

  3. (C)

    P and R only

  4. (D)

    P, Q and R only

Answer and solution

ANSWER

The correct answer is (B) P, R and S only.

Official answer: BSource: Official answer key, GATE 2020 BT (IIT Delhi)

  1. Setup: the plasmid has one continuous strand (ATCGCGATG) serving as template, and the complementary strand broken into two pieces at the same location: a left fragment ending in a free 3'-OH, then a gap of missing nucleotides, then a right fragment beginning with a free 5'-OH (not 5'-phosphate, as explicitly printed).

  2. Step 1 — Gap-filling needs DNA polymerase [R]: DNA polymerase extends from the available 3'-OH end (the left fragment's terminus), using the intact continuous strand as template, synthesizing the missing nucleotides across the gap until it abuts the right fragment. This converts the gap into a simple nick.

  3. Step 2 — Sealing that nick needs 5'-phosphate, so Polynucleotide kinase [S] is needed: the right fragment's 5' end is printed as 5'-OH, not 5'-phosphate. DNA ligase's mechanism requires a 5'-phosphate at the nick to form the phosphodiester bond; it cannot join a 5'-OH end. Polynucleotide kinase phosphorylates this 5'-OH to 5'-phosphate, making the nick ligatable.

  4. Step 3 — Sealing needs DNA ligase [P]: with a 3'-OH (from polymerase extension) apposed to a 5'-phosphate (from kinase action), DNA ligase forms the final phosphodiester bond, producing a covalently closed circular plasmid.

  5. Step 4 — Alkaline phosphatase [Q] is not required: this enzyme removes 5'-phosphate groups (its typical use is preventing vector self-ligation) — the opposite of what is needed here — so it plays no role and is excluded.

  6. Conclusion: the required enzymes are P (DNA ligase), R (DNA polymerase), and S (polynucleotide kinase) — option (B), 'P, R and S only'.

KEY IDEA

Closing a nicked/gapped circular plasmid needs, in order: (1) a polymerase to extend the available 3'-OH primer across the gap using the intact strand as template, (2) if the resulting nick's downstream terminus is 5'-OH rather than 5'-phosphate, a kinase to phosphorylate it (ligase cannot join a 5'-OH), and only then (3) ligase to seal the phosphodiester backbone into a covalently closed circle.

WATCH OUT FOR

Assuming DNA ligase alone (with polymerase) is sufficient to close any gap/nick, without checking the chemical nature of the 5' terminus; a 5'-OH end (as printed here) cannot be ligated until polynucleotide kinase converts it to 5'-phosphate. A related trap is confusing alkaline phosphatase (removes 5'-phosphates, used to prevent vector self-ligation) with polynucleotide kinase (adds 5'-phosphates) — they have opposite effects, so including alkaline phosphatase here is wrong.

From the GATE 2020 BT paper. Every question of this paper · Download the full paper · All 5 Libraries & cloning questions, with notes

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