1. 1
   In a replicating Escherichia coli chromosome, two replication forks move away from a single origin. At each fork, helicase opens the double helix. One new strand is made as a single continuous strand that grows towards the fork as it opens. The other new strand is made as a series of short sections, each about 1000–2000 nucleotides long, each synthesised in the direction away from the fork and later joined together.

   (a)

   How does DNA polymerase III add nucleotides to a growing strand?

   [1 mark]

   • AIt links the 5' end of each new nucleotide to the 3' end of the strand, so the strand grows in the 5' to 3' direction
   • BIt links the 3' end of each new nucleotide to the 5' end of the strand, so the strand grows in the 3' to 5' direction
   • CIt adds nucleotides to whichever end of the strand is nearer to the fork
   • DIt adds nucleotides to both ends of the strand at the same time

   (b)

   Why is one of the new strands made in short sections?

   [1 mark]

   • AHelicase can unwind only one of the two template strands at a time
   • BThe template strands are antiparallel and DNA polymerase can only synthesise 5' to 3', so on one template synthesis must run away from the fork
   • CPrimase cannot bind to the template of the continuous strand
   • DThe template of the discontinuous strand contains non-coding sequences that must be skipped

   (c)

   Outline the role of helicase at the fork and explain why an RNA primer is needed repeatedly on only one of the new strands.

   [2 marks]

   Total for question 1: 4 marks

2. 2
   Researchers compared three strains of E. coli. In strain 1 (normal), newly made DNA is almost entirely found in long molecules. In strain 2, which has one temperature-sensitive replication enzyme, cells kept at 42 °C accumulate large amounts of new DNA as fragments of about 1000–2000 nucleotides; these fragments contain no RNA. In strain 3, DNA polymerase III adds nucleotides at the normal rate but has lost its proofreading activity; the mutation rate of strain 3 is about 1000 times that of strain 1.

   (a)

   Which enzyme is most likely to be defective in strain 2 at 42 °C?

   [1 mark]

   • ADNA primase
   • BDNA polymerase I
   • CDNA ligase
   • DHelicase

   (b)

   What is the function of DNA polymerase I in a normal cell?

   [1 mark]

   • AIt synthesises RNA primers on the template strand
   • BIt adds most of the nucleotides to the leading strand
   • CIt joins adjacent Okazaki fragments together
   • DIt removes RNA primers and replaces them with DNA nucleotides

   (c)

   Explain why the mutation rate of strain 3 is much higher than that of strain 1.

   [2 marks]

   Total for question 2: 4 marks

3. 3
   A food-safety laboratory tests beef burgers for horse meat. DNA is extracted from each burger and PCR is run with two pairs of primers: one pair binds only to a sequence in horse DNA and produces a 227 bp fragment; the other binds only to a sequence in cattle DNA and produces a 271 bp fragment. The products are separated by gel electrophoresis alongside controls. Pure horse DNA gives a single band at 227 bp. Pure cattle DNA gives a single band at 271 bp. A tube containing all the reagents but water instead of DNA gives no bands. Burger 1 gives a band at 271 bp only. Burger 2 gives a strong band at 271 bp and a faint band at 227 bp.

   (a)

   Explain the roles of the primers and the temperature changes in producing the 227 bp fragment.

   [3 marks]

   (b)

   Evaluate the conclusion that burger 2 contains horse meat.

   [4 marks]

   Total for question 3: 7 marks

4. 4
   The E. coli chromosome is a circular DNA molecule of about 4.6 million base pairs. Replication begins at a single origin, and two replication forks move in opposite directions around the circle until they meet. Each fork moves at about 1000 nucleotides per second. Okazaki fragments in E. coli are about 1500 nucleotides long. Before proofreading, DNA polymerase III inserts an incorrect nucleotide about once in every 10⁵ nucleotides; after proofreading, the error rate is about once in every 10⁷ nucleotides.

   (a)

   Explain the differences between replication of the leading strand and the lagging strand, including the roles of the enzymes involved.

   [6 marks]

   (b)

   Using the data, discuss how E. coli copies its chromosome both quickly and accurately.

   [6 marks]

   Total for question 4: 12 marks

End of questions