RI Nov 16 H2 P3 ans

2016 H2 Biology P2 9744/03 1 (a) Describe the natural functions of these restriction enzymes. [2] 1. Used as a defense mechanism by bacteria against bacteriophage by cutting up foreign DNA, hence restricts multiplication of viruses; 2. Enzyme that recognizes and bind a specific 4-6 base pair DNA sequence called a restriction site* as its active site is complementary to the DNA sequence; 3. Enzyme cuts/breaks phosphodiester bonds* on specific positions on both DNA strands; (b) (ii) Use the information in Table 1.1 to com pare the effect of pH on immobilised P1 nuclease and free P1 nuclease. [4] 1. Compare peak + quote data The optimum pH for immobilized P1 nuclease was higher at pH 6.0 whereas free P1 nuclease peaked at pH 5.5; or The maximum nuclease activity for immobilized P1 nuclease was higher at 68 arbitrary units(a.u.), whereas maximum nuclease activity for free P1 nuclease was 65 arbitrary units. 2. Immobilised P1 nuclease activity was lower than free P1 nuclease from pH 5 to pH5.5. + quote data from either pH e.g. immoblised P1 nuclease acti vity was 49 a.u. whereas free P1 nuclease was 56 a.u. at pH 5.0, or immoblised p1 nuclease activity was 7 a.u. lower than free P1 nuclease at pH 5.0 3. Immobilised P1 nuclease activity was higher than free P1 nuclease from pH6.0 to pH7.0. + quote data from any 1 pH e.g. immoblised P1 nuclease acti vity was 68 a.u. whereas free P1 nuclease was 48 a.u. at pH 6.0, or immoblised p1 nuclease activity was 20 a.u. higher than free P1 nuclease at pH 6.0); 4. Both had similar trend of increasing in activity from pH4.5 to pH5.5 then decreasing from pH6.0 to pH7.0 + quote data (ii) With reference to your knowledge of enzyme structure and the information in (b), explain the higher activity of immob ilised P1 nuclease, compared to free P1 nuclease, at temperatures above 30 °C, as shown in Table 1.2. [3] 1. At higher temperatures, intramolecular vibrations increase, which results in the breaking of bonds that determine the conformation of the enzyme in the free P1 nuclease. 2. Resulting in the denaturation of the enzyme due to a change in conformation of the enzyme active site, decreasing the ra te of enzyme activity of free P1 nuclease 3. Binding of P1 nuclease to matrix, st abilize the enzyme’s structure at higher temperatures, reducing intramolecular vibrations, prevents breaking of bonds, and retaining the conformation of enzyme active site. 4. Activity of free P1 nuclease starts to drop at 60°C which is lower than that of 70°C in immobolised P1 nuclease. (ii) Suggest why P1 nuclease was needed in the production of this particular recombinant DNA molecule. [3] 1. P1 nuclease was needed to break down the different sticky ends produced by the digestion of HindIII and EcoRI. 2. As it is able to break down single- stranded DNA, leaving the double stranded DNA fragment intact.

3. Scientist wil