Lecture 13

Question 1

In what phase of the cell cycle does the cell replicate its DNA?

Answer 1

In the S phase

Question 2

Which of the DNA polymerase are involved in DNA replication?

Answer 2

DNA polymerase 1 and 3

Question 3

Which polymerase is part of a large multiunit complex in eukaryotic DNA replication?

Answer 3

BnB

Question 4

What group is at the 3’ end of the DNA, and what group does it attach to in the incoming dNTP? What molecule is released from the reaction?

Answer 4

The pentose sugar is at the 3’ end of the DNA. It attaches to one of the phosphate groups on the incoming dNTP which release the other two phosphate groups.

Question 5

What ions are an essential part of the catalytic site in DNA polymerase, what amino acids are coordinating them, and what do the ions do?

Answer 5

Magnesium ions are an essential part of the catalytic site of DNA polymerase. Aspartic acid coordinates the Mg2+ to interact with the phosphate groups on the incoming dNTP.

Question 6

Considering DNA polymerase I and III, which inserts more bases in a row? Which has 3’ -> 5’ exonuclease activity? Which has 5’-> 3’ exonuclease activity?

Answer 6

DNA polymerase III inserts more bases in a row. Both DNA pol I and III have 3’ -> 5’ exonuclease while only DNA pol I hate 5’ -> 3’ exonuclease.

Question 7

What is exonuclease activity, and in carrying out what functions do the polymerases use their exonuclease activity?

Answer 7

Exonuclease activity is the cleaving off of nucleotides from a polynucleotide chain. The polymerases use this function when proof reading the DNA sequence.

Question 8

What is an origin of replication?

Answer 8

The origin of replication is a particular sequence of DNA which replication is initiated.

Question 9

What is a replication bubble?

Answer 9

A replication bubble is a the unwinding of the parent DNA from two replication forks that are close to each other, running in opposite directions forming a ‘bubble’.

Question 10

What is a replication fork?

Answer 10

A replication fork is where the parent DNA is unwounded and the single strands are quickly replicated.

Question 11

The bacterial origin of replication has an AT-rich region. What is the significance if this?

Answer 11

The base pairing between A and T are weaker than G and C. An AT rich region is easier to separate than a GC rich region.

Question 12

Ligase

Answer 12

Ligase is an specific enzyme that facilitates in the joining of DNA strands together by catalysing the formation of phosphodiester bonds.

Question 13

Primase

Answer 13

Primase is an enzyme that catalyses the synthesises short strands of RNA sequences called primers.

Question 14

Helicase

Answer 14

Helicase binds to the DNA and stimulates the separation of the two strands.

Question 15

Topoisomerase (gyrase)

Answer 15

This enzyme facilitates in the negative supercoiling of the DNA which relieves the tension in the DNA.

Question 16

Sliding clamps

Answer 16

It is a protein fold that attaches to the DNA polymerase and prevents it from dissociating from the DNA strands.

Question 17

Clamp loader

Answer 17

The clamp loader is a complex that places a beta clamp onto a new template primer allowing the Okazaki fragments to be synthesised on the lagging strand.

Question 18

Single-stranded binding proteins

Answer 18

They are proteins that bind to the single strands of DNA being unwind and protects it from being nuclease while keeping the strands separated.

Question 19

What is the primer in DNA replication made of and what is it for? Why can’t the strand of DNA just be replicated without a primer?

Answer 19

The primer in DNA replication is made up of RNA nucleotides. Since DNA polymerase can only work on double stranded template, RNA primers are need for the synthesis of the new DNA strand to take place.

Question 20

What is the difference between leading and lagging strand synthesis?

Answer 20

The synthesis of DNA on the leading strand is continuous while it is discontinuous on the lagging strand. Okazaki fragments are produce as a by product of this discontinuity.
The lagging strand requires DNA ligase and many RNA primers whereas the leading strand does not require DNA ligase and has one RNA primer.

Question 21

What is an Okazaki fragment?

Answer 21

Okazaki fragments are short single strands of DNA formed on the lagging strand.

Question 22

What protein removes the primer?

Answer 22

DNA polymerase I removes the RNA primers and replaces them with DNA nucleotides. (5’ -> 3’ exonuclease)

Question 23

How many molecules of DNA polymerase III are in the replication machinery at a replication fork?

Answer 23

Two molecules of DNA polymerase III are needed; one on the leading strand and one of the lagging strand.

Question 24

What are mechanisms involving DNA polymerase to prevent the insertion of incorrect nucleotides?

Answer 24

DNA polymerase is able to proof read the newly synthesised sequence. It has exonuclease activity that allows it to remove incorrect nucleotides and fix it. The binding of the base pairs also contributes to preventing the insertion of incorrect nucleotides. AT and GC fit together perfectly while AC and GT would cause are bulge in the double strand of DNA.

Question 25

Does DNA polymerase use 5’ -> 3’ exonuclease activity or 3’ -> 5’ exonuclease activity to fix incorrect nucleotides?

Answer 25

The DNA polymerase uses 3’ to 5’ exonuclease activity to fix incorrect nucleotides.

Question 26

When a nucleotide is removed by this process because it was incorrect, can it then be added in the next correct position? Explain why or why not.

Answer 26

The new nucleotide would be added the next correct position. This is because the DNA polymerase coordinates with the 3’ of the sugar before it to connect the new nucleotide to the strand. This prevents the nucleotide from attaching to the wrong position.

Question 27

What does it mean that DNA replication is semi-conservative?

Answer 27

DNA replication is semi-conservative as it conserves half of the parent DNA when replicated. The new daughter helices have one strand from the parent helix and one newly synthesised strand.

Question 28

What is a telomere, and what does telomerase do?

Answer 28

A telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration. Telomerase is a reverse transcriptase enzyme that uses its own RNA molecule to elongate telomeres.

Question 29

What would happen if telomerase did not function in your cells?

Answer 29

If telomerase did not function properly, the DNA strands would get shorter with each DNA replication, reducing the amount of telomeres until all is gone. It would lead to the loss of genetic material.

Question 30

Why do prokaryotes not need telomeres?

Answer 30

Prokaryotes do not need telomeres as their DNA is circular and thus the end of the chromosome does not degrade during replication.