Questions on DNA replication

Question 1

Describe how a molecule of DNA is copied semi-conservatively, naming the enzymes involved and the bonds that are broken and formed.

Accepted Answer

B1 DNA helicase breaks the hydrogen bonds between complementary bases, so the two strands separate and the helix unwinds B1 each of the two separated strands acts as a template B1 free DNA nucleotides align against the exposed bases by complementary base pairing, adenine to thymine and cytosine to guanine B1 DNA polymerase catalyses the condensation reactions that join the aligned nucleotides, forming phosphodiester bonds along the new backbone A1 each daughter molecule ends up with one original strand and one newly synthesised strand, which is what semi-conservative means

Question 2

Explain why one of the two new DNA strands is built continuously while the other is built as a series of short fragments.

Accepted Answer

B1 DNA polymerase can only attach a nucleotide to a free 3′ hydroxyl group, so it can only build a strand in the 5′ to 3′ direction B1 the two template strands are antiparallel, so the enzyme cannot travel the same way along both of them B1 on one template it moves in the direction the replication fork is opening, producing one continuous leading strand B1 on the other it must work away from the fork, so it waits for template to be exposed and copies a short piece backwards each time, giving the Okazaki fragments of the lagging strand

Question 3

Describe how detergent and salt are used to release DNA from tissue during a DNA extraction practical, and describe the specific job of each.

Accepted Answer

B1 the tissue is mashed and mixed with a warm solution of detergent and salt B1 the detergent disrupts the phospholipid bilayers of the cell surface membrane and the nuclear envelope, since a detergent dissolves lipid, which releases the DNA into solution B1 the sodium ions of the salt are attracted to and shield the negatively charged phosphate groups of the DNA backbone B1 shielding those charges stops the DNA molecules repelling one another, so they can later clump together rather than staying dispersed

Question 4

Explain how the result of the second generation of the Meselson-Stahl experiment eliminated the dispersive model of replication, once the conservative model had already been ruled out.

Accepted Answer

B1 the dispersive model predicts that every molecule, in every generation, is an even patchwork of old and new sequence, so after the second generation it predicts a single band, this time three quarters of the way toward the light position, rather than two separate bands B1 the semi-conservative model instead predicts two distinct bands after the second generation: half the molecules still contain one original heavy strand and remain intermediate, while the other half are now entirely light B1 two distinct bands were observed rather than one shifted band, which matches the semi-conservative prediction and not the dispersive one B1 only after the second generation do the semi-conservative and dispersive models make different, testable predictions

Question 5

A lagging strand 1500 nucleotides long is synthesised as Okazaki fragments each approximately 150 nucleotides long. Calculate the number of fragments produced, and calculate the number of phosphodiester bonds ligase must form to join them into one continuous strand.

Accepted Answer

M1 number of fragments = total length ÷ fragment length = 1500 ÷ 150 A1 10 Okazaki fragments M1 joining n fragments into one continuous strand needs n − 1 new bonds, since each join is between one fragment and the next A1 ligase must form 10 − 1 = 9 phosphodiester bonds

Question 6

A student says that replication copies just one strand of the original molecule, so you end up with the old molecule intact and a brand new molecule made entirely from scratch beside it. Discuss whether this is an accurate description of semi-conservative replication.

Accepted Answer

B1 what is right: the student is correct that one new molecule is produced from the old one, and that new material is synthesised during replication B1 against: this description is actually the conservative model, which predicts that the original double helix stays intact and an entirely new one is built alongside it; it is not what semi-conservative replication does B1 against: in semi-conservative replication both original strands separate and each acts as a template, so both of the two daughter molecules contain one original strand and one newly synthesised strand, rather than one molecule being entirely old and the other entirely new B1 judgement: no molecule after replication is entirely old; the Meselson-Stahl experiment's single intermediate band after one generation is exactly the evidence against this description

Question 7

Bacteria whose DNA contains only ¹⁵N are transferred to a medium containing only ¹⁴N and allowed to divide four times. Calculate the percentage of the DNA molecules that still contain a ¹⁵N strand.

Accepted Answer

M1 the two original heavy strands are never destroyed, so exactly two molecules contain one M1 after four divisions there are 2⁴ = 16 molecules altogether, so the proportion is 2 ÷ 16 A1 12.5 per cent of the molecules contain a ¹⁵N strand, the remaining 87.5 per cent being entirely light

Question 8

DNA polymerase corrects most of its own copying errors, but a few substitutions survive in every cell division. Suggest three reasons why such a substitution often has no effect on the organism.

Accepted Answer

B1 the substitution may fall in a non-coding stretch of DNA, so no polypeptide is affected by it B1 the altered triplet may code for the same amino acid as the original, so the polypeptide is unchanged B1 the amino acid may change in a region of the protein where the substitution makes no difference to the folded shape, so the protein still functions

Question 9

In the Meselson and Stahl experiment, a single band of intermediate density appeared after one generation in the light medium. Explain why this ruled out the conservative model of replication but could not rule out the dispersive model.

Accepted Answer

B1 the conservative model never puts old and new material in the same molecule, so it predicts one heavy band and one light band and no intermediate band at all B1 one intermediate band and no heavy band is therefore impossible under the conservative model, which is ruled out B1 a dispersive patchwork of half-old and half-new sequence would also settle at intermediate density after one generation, so the two remaining models predict the same first-generation result and only the second generation separates them

Question 10

Explain why a protease is added to a DNA extract before it is precipitated, and explain why the ethanol used must be ice-cold.

Accepted Answer

B1 in a eukaryotic nucleus DNA is wound around histone proteins, and the extract also contains other protein besides, so a protease is added to hydrolyse this protein and leave the DNA free B1 DNA is less soluble in cold ethanol than in warm ethanol, so using ice-cold ethanol makes more of the DNA come out of solution as visible fibres B1 low temperature also slows any enzymes remaining in the extract that would otherwise break the DNA molecules up before they can be spooled out

Question 11

Explain how a single base substitution in the gene for the β-globin chain of haemoglobin causes sickle-cell anaemia.

Accepted Answer

B1 a single base substitution changes one triplet in the gene, so the corresponding codon in the mRNA changes B1 that changed codon specifies a different amino acid, valine in place of glutamic acid, at one position in the polypeptide B1 the altered haemoglobin this produces polymerises at low oxygen concentrations, and that distorts the shape of the red blood cell containing it

Question 12

State the role of DNA helicase and state the role of DNA ligase in the replication of a DNA molecule.

Accepted Answer

B1 helicase breaks the hydrogen bonds between the paired bases, so the two strands separate and the helix unwinds B1 ligase joins the Okazaki fragments of the lagging strand into one continuous strand, forming the phosphodiester bonds between them

Question 13

After proofreading and mismatch repair, replication leaves about one uncorrected error per billion base pairs copied. The human genome is about 3.1 × 10⁹ base pairs. Calculate the expected number of new errors each time one copy of the genome is made.

Accepted Answer

M1 divide the base pairs copied by the error interval: 3.1 × 10⁹ ÷ 1 × 10⁹ A1 about 3 new errors per copy, accepting 3.1

Question 14

Give the type of bond broken by DNA helicase, and give the type of bond formed by DNA polymerase.

Accepted Answer

B1 helicase breaks the hydrogen bonds between paired bases, across the molecule B1 DNA polymerase forms phosphodiester bonds, which are covalent, along the backbone of the new strand

Question 15

DNA polymerase cannot start a new strand on its own: it can only add nucleotides to the end of a strand that already exists, so each new stretch of DNA is begun on a short RNA primer made by another enzyme. At a replication fork one new strand is built continuously and the other as a series of short fragments. Explain why the strand built in fragments needs many primers, while the strand built continuously needs only one.

Accepted Answer

B1 each fragment is a separate stretch of DNA begun from nothing, and polymerase cannot begin one without an existing strand to extend, so every fragment needs its own primer B1 the continuous strand is extended without a break from its single starting point as the fork opens, so after the first primer the polymerase always has the end of the growing strand to add to