DNA Replication

Question 1

What complexes recognise the replication origins?

Answer 1

Pre-replication complexes

Question 2

What happens at the replication bubble?

Answer 2

At the replication fork, one parental DNA molecules splits into two daughters.

Question 3

What stage of the cell cycle does replication happen?

Answer 3

DNA replication occurs in the S phase of the cell cycle

Because there 3 billion base pairs, there multiple replication sites in one cell.

Question 4

What happens when two replication forks meet?

Answer 4

They coealesce

Question 5

How long is the bacteria cell cycle and what phases does it consist of?

Answer 5

Happens in 20-30 minutes which consists of M phase and S phase

Question 6

Why does bacteria have only one replication origin?

Answer 6

Their DNA is circular but eukaryotic cells have multiple replication origins to make synthesis quicker since their DNA is linear.

Question 7

How many DNA polymerases do bacteria and eykaryotic cells have?

Answer 7

Bacteria- 5Eukaryotic- up to 17

Question 8

Recall the five well known DNA polymerases and their functions

Answer 8

· Alpha and beta - replication
· Delta &epsilon - cause elongation in replication
· Gamma – mitochondrion DNA replication

Question 9

Summarise the requirements of DNA polymerase(4)

Answer 9

Requires a DNA template,a DNA or RNA primer,the four deoxyribonucleoside triphosphate building blocksMg2+

Question 10

Describe the process of replication (7)

Answer 10

“1. DNA helicase first unwinds the double helix by breaking the hydrogen bonds using ATP,

2. it creates extra supercoils (negative supercoiling) and topoisomerase II (in bacteria this is called DNA gyrase) relieves the stress and tension created from the -ve supercoils by adding +ve supercoils.
3. SSBPs bind to them (single stranded binding proteins) which help keep the strands apart so they don’t reanneal, and they help protect them from degradation by nucleases.
4. a primer is laid down by RNA primase at the 5’ ends of both strands where the replication fork has formed
5. The leading strand only needs one primer as the synthesis is continuous
6. the lagging strand needs more than one primer as the synthesis is discontinuous because it copies away from the replication fork as it opens
7. the okazaki fragments are joined by DNA ligase”

Question 11

Explain the differences between the lagging strand and leading strand?

Answer 11

The leading strand only needs one primer as the synthesis is continuous but the lagging strand needs more than one primer because it copies away from the replication fork so as the strand opens, the polymerase needs to ‘go back’ and start from the replication fork.

Question 12

Name thedeoxyribonucleoside triphosphates

Answer 12

dATP, dCTP, dGTP, dTTP

Question 13

Define a primer and how are they synthesised?(2)

Answer 13

1. A primer is a short RNAsequence that provides a starting point for DNA synthesis.
2. A primer must be synthesized by primase, which is a type of RNA polymerase

Question 14

Why is Mg2+ needed with polymerase?(2)

Answer 14

1. One of the magnesium ions draws the proton away from the 3 ′ hydroxyl group of the terminal nucleotide of the primer, facilitating the nucleophilic attack of the negatively charged 3 ′ oxygen atom on the phosphate of the incoming nucleoside triphosphate.
2. The second magnesium ion stabilizes the pyrophosphate, promoting its release.

Question 15

Why are new nucleotides added to the 3’ prime end?

Answer 15

During the polymerization reaction, the —OH group at the 3 ′ end of the primer carries out a nucleophilic attack on the 5 ′ ‐phosphate of the incoming nucleoside triphosphate.

Question 16

Describe the nucleophillic attack between the triophosphates and the growing strand(2)

Answer 16

1. the release of pyrophosphate- Pyrophosphate is a diphosphate (PPi ), by pyrophosphotase.
2. This happens when a covalent bond is formed between the 3’ oxygen of the growing polynucleotide and the α-phosphate of the incoming nucleotide.

Question 17

How are primers made?

Answer 17

Uses ribonucleoside triphosphates to synthesize short RNA primers on the lagging strand In eucaryotes, these primers are about 10 nucleotides long and are made at intervals of 100–200 nucleotides on the lagging strand.

Question 18

How are the breaks between Okazaki fragments sealed?

Answer 18

An enzyme DNA ligase that links these fragments

Question 19

What is the role of DNA gyrase in super coiling in bacteria?

Answer 19

1. DNA gyrase molecules travel along the DNA ahead of the replication fork, removing positive supercoils.
2. a process that is driven by the energy released during ATP hydrolysis

Question 20

What is the difference between topoisomerase I and Type II topoisomerase

Answer 20

1. Type I topoisomerases relax DNA (i.e., remove supercoils) by nicking and closing one strand of duplex DNA.
2. Type II topoisomerases change DNA topology by breaking and rejoining double-stranded DNA(gyrase)

Question 21

How long are pirmers?

Answer 21

10 nucleotides

Question 22

What is the role of B clamps in stopping the dissociation of polymerase from the strand after short synthesise?(2)

Answer 22

1. keeps the polymerase firmly on the DNA when its is moving.
2. Assembly around the helix require ATP by a special protein complex- the clamp loader

Question 23

What is the difference between the tightness of the clamp-polymerase relationship in leading and lagging strand?(2)

Answer 23

1. On the leading-strand template, the moving DNA polymerase is tightly bound to the clamp, and the two remain associated for a very long time.
2. However, on the lagging-strand template, each time the polymerase reaches the 5′ end of the preceding Okazaki fragment, the polymerase is released

Question 24

Why is a mismatched pair a problem?

Answer 24

A mismatched base pair causes a distortion in the geometry of the double helix

Question 25

How does DNA replication proceed with such high fidelity?(3)

Answer 25

1. Base pairing in DNA 2 .Proof-reading by DNA polymerase 3. Mismatch repair system- proteins patrol the newly synthesised strand.

Question 26

How does the mismatch repair gene correct changes using example of e.coli?(2)

Answer 26

1. The enzyme recognize the member of the mismatched pair by the nucleotide from the parental strand which contains the correct nucleotide.  2. In E. coli , the two strands are distinguished by the presence of methylated adenosine residues on the parental strand.

Question 27

Recall the mutations that can occur bacteria rpair genes

Answer 27

mutH, mutL, mutS. Resulting in colon cancers. 

Question 28

Why are repliaction inhibitors important?

Answer 28

Replication inhibitors are important because they are antibacterial, antitumour and antiviral agents. 

Question 29

What is the error rate in human DNA replication?

Answer 29

" | 1 in 1010 with a replication rate of 3x109"

Question 30

How is the single strand of DNA protected and prevented from resealing when it is opened up? (3)

Answer 30

1. By binding to to binding protein.  2. These proteins bind selectively to single stranded DNA, keeping it in an extended state  3. preventing it from becoming rewound or damaged.

Question 31

How does ATP help helicase to separate stands?(2)

Answer 31

1. The hydrolysis of ATP can change the shape of a protein molecule in a cyclical manner that allows the protein to perform mechanical work. 2. DNA helicases use this principle to propel themselves rapidly along a DNA single strand. 

Question 32

How does the polymerase III molecule move from one site on the replication strand template strand to another site that is closer to the replication fork?

Answer 32

The two tethered polymerases can replicate both strands by looping the DNA of the lagging‐strand template back on itself, causing this template to have the same orientation as the leading‐strand template. Trombone model