Week 1 (DNA replication)

Question 1

What is DNA replication?

Answer 1

The accurate duplication of vast quantities of genetic information carried in chemical form as DNA

Question 2

What is DNA templating?

Answer 2

The mechanism the cell uses to copy the nucleotide sequence of one DNA strand into a complementary DNA sequence.

Question 3

What is the function of DNA helicase?

Answer 3

Unwinds DNA and exposes the hydrogen bond-donor and acceptor groups on each DNA base for base-pairing with the appropriate nucleotide

Question 4

What is the function of DNA polymerase?

Answer 4

Polymerises free nucleotides- dNTPS’s (deoxyribonucleoside triphosphates) into DNA via a single strand at the 3’ OH end of a polynucleotide chain

Question 5

What is the fundamental reaction by which DNA is synthesised?

Answer 5

• The addition of a deoxyribonucleotide to the 3’ end of a polynucleotide chain (primer strand)
• Base-pairing between an incoming deoxyribonucleoside triphosphate and an existing stand of DNA (template strand) guides the formation of a new DNA strand with a complementary nucleotide sequence

Question 6

What direction does DNA polymerase synthesise DNA?

Answer 6

5’-3’ direction

Question 7

What direction does DNA polymerase read DNA template strand?

Answer 7

3’-5’ direction

Question 8

What does the energy to power the reaction come from?

Answer 8

The hydrolysis of the triphosphate (3P) to two molecules pyrophosphate (inorganic phosphate)

Question 9

What initiates nucleotide addition reaction?

Answer 9

The proper base-pair geometry of a correct incoming deoxyribonucleoside triphosphate causes the polymerase to tighten around the base pair , thereby initiating the nucleotide addition reaction

Question 10

What is the DNA replication fork?

Answer 10

• A localised region of replication that moves progressively along the parental DNA double helix.
• Because of its Y-shaped structure, this active region is called a replication fork

Question 11

What happens at the replication fork?

Answer 11

A multienzyme complex that contains the DNA polymerase synthesise the DNA of both new daughter strands

Question 12

What is the leading strand?

Answer 12

The DNA daughter strand that is synthesised continuously

Question 13

What is the lagging strand?

Answer 13

• The DNA daughter strand that is synthesised discontinuously
• The direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth

Question 14

Why are okazaki fragments created?

Answer 14

• DNA on the lagging strand is also polymerised in the 5’ to the 3’ direction
• The DNA synthesised on the lagging strand must be made initially as a series of short DNA molecules, called Okazaki fragments
• Okazaki fragments are synthesised sequentially, with those nearest the fork being the most recently made

Question 15

What is the function of DNA primase?

Answer 15

• The enzyme that synthesises the short RNA primers made on the lagging strand using DNA as a template
• The enzyme can start a new polypeptide chain by joining two nucleoside triphosphates together

Question 16

When is a primer needed for the leading strand?

Answer 16

At the start of replication

Question 17

When is a primer needed for the lagging strand?

Answer 17

Each time the DNA polymerase completes a short DNA Okazaki fragment, it must start synthesising a completely new fragment at a site further along the template strand

Question 18

What does primase use to synthesise short RNA primers?

Answer 18

Ribonucleotide triphosphates

• Usually 10 nucleotides long
• Made at intervals of 100-200 nucleotides on the lagging strand

Question 19

When does the synthesis of each okazaki fragment end?

Answer 19

When the DNA polymerase runs into the RNA primer attached to the 5’ end of the previous fragment

Question 20

What is the function of DNA ligase?

Answer 20

Joins the 3’ end of the new DNA fragment to the 5’ end of the previous one to complete the process therefore producing a continuous DNA chain
(seals a broken phosphodiester bond using energy from ATP)

Question 21

Why might an erasable RNA primer be preferred to a DNA primer?

Answer 21

More efficient self-correction

Question 22

What is the function of Single stranded binding proteins (SSBs) ?
(Helix destabilizing proteins)

Answer 22

Bind tightly to exposed single strand DNA without covering the bases, they aid helicases by stabilizing the unwound strand and preventing annealing

Question 23

Why is it useful for DNA polymerase molecules to dissociate quickly from a DNA molecule?

Answer 23

Allows a DNA polymerase molecule that has just finished synthesizing one Okazaki fragment on the lagging strand to be recycled quickly, so as to begin the synthesis of the next Okazaki fragment on the same strand

Question 24

Why is it not useful for DNA polymerase molecules to dissociate quickly from a DNA molecule?

Answer 24

The rapid dissociation would make it difficult for the polymerase to synthesise the long DNA strands produced at a replication fork

Question 25

What is the function of the accessory protein PCNA?

Answer 25

Functions as a regulated sliding clamp that keeps the polymerase firmly on the DNA as it is moving but releases as soon as the polymerase runs into a double stranded region of DNA

Question 26

What is the function of DNA topoisomerase?

Answer 26

Relieves over winding of DNA ahead of the replication
fork
Breaks phosphodiester linkage in one DNA strand so the two ends of the double helix can rotate relative to each other, relieving accumulated strain

Question 27

What is the function of topoisomerase I

Answer 27

Produces a transient single strand break in the phosphodiester backbone allows the two sections of DNA helix on either side of the nick to rotate freely relative to each other

Question 28

What is the function of topoisomerase II?

Answer 28

Forms a covalent linkage to both strands of the DNA helix at the same time making a transient double-strand break in the helix
They also hydrolyse ATP

Question 29

Topoisomerase II uses ATP hydrolysis to perform the following set of reactions efficiently:

Answer 29

(1) It breaks one double helix reversibly to create DNA ‘gate’
(2) It causes the second nearby double helix to pass through the this opening
(3) It then reseals the break and dissociates from the DNA

At crossover points generated by supercoiling, passage of the double helix through the gate occurs in the direction that would reduce supercoiling

Question 30

Give a summary of DNA replication

Answer 30

• DNA replication takes place at a Y-shaped structure called a replication fork
• A self correcting DNA polymerase enzyme catalyses nucleotide polymerisation in a 5’-3’ direction, copying a DNA template strand with remarkable fidelity
• Since the two strands of a DNA double helix are antiparallel, this 5’-3’ DNA synthesis can take place continuously on only of the strands at a replication fork (the leading strand)
• On the lagging strand, short DNA fragments must be made by a “backstitching” process
• Because the self-correcting DNA polymerase cannot start a new chain, these lagging-strand DNA fragments are primed by short RNA primer molecules that are subsequently erased and replaced with DNA

Question 31

Give a summary of DNA replication and the proteins involved

Answer 31

(1) DNA polymerase and DNA primase to catalyse nucleoside triphosphate polymerization
(2) DNA helicases and single-strand DNA-binding (SSB) proteins to help in opening up the DNA helix so it can be copied
(3) DNA ligase and an enzyme that degrades RNA primers to seal together the discontinuously synthesised lagging strand
(4) DNA topoisomerases to help relieve helical winding and DNA tangling problems
- Many of these proteins associate with each other at a replication fork to form a highly efficient “replication machine”

Question 32

What is supercoiling?

Answer 32

DNA double helix twists around itself

Question 33

What amino acid is at the active site of topoisomerase I?

Answer 33

Tyrosine