23. In a cell free extract containing DNA polymerase I, Mg²⁺, dATP, dGTP, dCTP and dTTP³H), the following DNA

molecules were added:

1. Single stranded closed circular DNA moleculecontaining 824 nucleotides.
2. Single stranded closed circular DNA molecule having1578 nucleotides base paired with a linear singlestandard DNA molecule of 824 nucleotides having a

free-3′-OH group.

1. Double stranded linear DNA molecule containing 1578 nucleotides having free-3’OH group at both ends.
2. Double stranded closed circular DNA molecule having 824 nucleotides.

The rate of DNA synthesis was measured by

incorporation of thymidine in the DNA molecule and expressed as the percentage of DNA synthesis relative to total DNA input. Which one of the following graphs represents the correct result?

Introduction

DNA Polymerase I (Pol I) from E. coli is a multifunctional enzyme involved primarily in processing Okazaki fragments during DNA replication. It has polymerase, 3′→5′ exonuclease (proofreading), and 5′→3′ exonuclease activities. Understanding how Pol I acts on different DNA substrates in vitro helps elucidate its biological function and substrate specificity.

In a cell-free extract containing DNA Polymerase I, Mg^2+, and the four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, and tritiated dTTP), DNA synthesis can be measured by incorporation of labeled thymidine into DNA molecules. The substrates tested include:

• (a) Single-stranded closed circular DNA (824 nucleotides)

• (b) Single-stranded closed circular DNA (1578 nucleotides) hybridized with a linear single-stranded DNA (824 nucleotides) having a free 3′-OH group

• (c) Double-stranded linear DNA (1578 nucleotides) with free 3′-OH ends

• (d) Double-stranded closed circular DNA (824 nucleotides)

Expected DNA Synthesis Activity on Each Substrate

1. (a) Single-stranded closed circular DNA (ssDNA, 824 nt)

   • Pol I requires a primer with a free 3′-OH to initiate DNA synthesis.

   • Pure ssDNA without a primer or free 3′-OH end will not support significant DNA synthesis.

   • Therefore, DNA synthesis on this substrate will be very low or negligible.

2. (b) Hybrid molecule: ssDNA closed circular (1578 nt) base-paired with linear ssDNA (824 nt) with free 3′-OH

   • The linear strand provides a primer-template junction with a free 3′-OH end.

   • This substrate mimics a primed template and is efficiently extended by Pol I, leading to significant DNA synthesis.

   • This substrate will show the highest DNA synthesis activity relative to others.

3. (c) Double-stranded linear DNA (1578 nt) with free 3′-OH ends

   • Pol I can extend from free 3′-OH ends on linear dsDNA.

   • DNA synthesis will occur but may be less efficient compared to a primed ssDNA template due to lack of processivity and potential end effects.

   • Moderate DNA synthesis is expected.

4. (d) Double-stranded closed circular DNA (824 nt)

   • Pol I cannot initiate DNA synthesis on fully double-stranded closed circular DNA without a primer or nick.

   • Unless the DNA contains nicks or gaps, DNA synthesis will be minimal or absent.

   • Therefore, low DNA synthesis is expected.

Summary of Expected DNA Synthesis Rates (Relative to DNA Input)

Explanation

• DNA Polymerase I requires a primer-template junction with a free 3′-OH group to add nucleotides.

• Substrate (b) provides an ideal primed template, allowing efficient DNA synthesis.

• Linear dsDNA with free 3′-OH ends (c) allows extension but may be less efficient due to lack of processivity.

• Pure ssDNA without primers (a) and closed circular dsDNA without nicks (d) do not provide initiation sites for Pol I, resulting in minimal synthesis.

Conclusion

In a cell-free system with DNA Polymerase I and required dNTPs, the hybrid DNA substrate (b) containing a primed template with a free 3′-OH end will exhibit the highest DNA synthesis. Linear dsDNA with free 3′-OH ends (c) will show moderate synthesis, while single-stranded closed circular DNA (a) and double-stranded closed circular DNA (d) will show little to no DNA synthesis.