Lecture 5- DNA replication

Question 1

Which direction does DNA synthesis occur in?

Answer 1

Occurs on a 5’ to 3’ prime direction mediated by the formation of a phosphodiester bonds.

Question 2

What happens in the chemical reaction for DNA replication?

Answer 2

1. The 3’ hydroxyl at the end of the growing DNA strand makes a nucleophile attack on the phosphate of the incoming dNTP
2. This forms a diester bond
3. Therefore the nucleotide is incorporated and the pyrophosphate is released

Question 3

Describe DNA synthesis via a replication fork

Answer 3

1. DNA helicase separates the double stranded DNA
2. This enables a primer to form on the leading strand with DNA synthesis occurring in 5’ to 3’ direction in this DNA replication fork
3. On the lagging strand, DNA synthesis still occurs 5’ to 3’ but in the opposite direction to the leading strand
4. The antiparallel orientation of parental strands and unidirectional orientation of new DNA synthesis means that both new strands cannot be synthesised continuously
5. To facilitate the lagging strand, Okazaki fragments produced by the lagging strand
6. To keep up with the leading strand, the lagging strand continuously synthesised short DNA fragments

Question 4

What type and which direction is the synthesis of the leading and lagging strand?

Answer 4

Both occur 5’ to 3’

Leading strand synthesis is continuous, lagging strand synthesis is discontinuous

Question 5

What is the job of DNA polymerase?

Answer 5

Use free -OH groups to add nucleotides to the 3’ ends of existing DNA strands

Question 6

How is a RNA primer made?

Answer 6

The short RNA primer is synthesised using template and NTPs by DNA primase

Question 7

Outline the steps on synthesising DNA chains

Answer 7

1. DNA primase makes RNA primer
2. Primase dissociates from the DNA template leaving a primer-template junction
3. DNA polymerase extends the RNA primer from the primer-template junction
4. The RNA primer are removed by ribonuclease H
5. The gap that is left is filled by DNA polymerase and the nick is sealed by DNA ligase

Question 8

What does DNA helicase do?

Answer 8

Using ATP to separate parental DNA strands by breaking H bonds

Question 9

Give 2 diseases causes by mutation in genes encoding DNA helicase?

Answer 9

1. Werner Syndrome

2. Bloom Syndrome

Question 10

What increases the processivity of DNA polymerases?

Answer 10

The association with a sliding clamp

Question 11

What is processivity?

Answer 11

An enzymes ability to catalyse consecutive reactions without releasing its substrate

Question 12

Explain the function of proceessive DNA polymerases?

Answer 12

• Add multiple nucleotides increasing the rate of DNA synthesis
• DNA polymerase’s ability to slide along the DNA template increased processivity

Question 13

What is the role of the clamp loader?

Answer 13

The clamp loader used ATP hydrolysis to load the sliding clamp onto the DNA near the primer-template junction

Question 14

What is the role of the sliding clamp?

Answer 14

1. The sliding clamp is locked onto the DNA which causes the displacement of primase and triggers the recruitment of DNA polymerase.
2. DNA is then reeled through the replisome and polymerase synthesised DNA continuously on the leading strand
3. On the lagging strand, DNA polymerases and synthesises DNA in bursts

Question 15

What do SSBs do?

Answer 15

Expose single stranded DNA in the replication fork, making it available for templating synthesis of the new DNA strand and stopping the formation of DNA hairpins

Question 16

How do DNA hairpins form?

Answer 16

The leading and lagging stands can anneal back on itself due to complementary DNA, creating hairpins

Question 17

Without SSBs, DNA hairpins can form. What is the problem with this?

Answer 17

Hairpins impede the progress of DNA polymerase and compromise its processivity.

Question 18

What is the role of DNA topoisomerases?

Answer 18

Prevent DNA from becoming tangled during DNA replication and enhancing the processivity of DNA polymerase

Question 19

How does DNA topoisomerases work?

Answer 19

Relieve tension by transiently nicking and resealing the parental double stranded DNA helix. This prevents DNA from becoming tangled which keeps the replication fork open for maximal processivity of DNA polymerases

Question 20

Where does replication begin in E.coli?

Answer 20

Replication begins at a genetically defined replicator site called oriC

Question 21

How is DNA replication initiated?

Answer 21

1. An initiator protein binds to oriC and promotes the melting of the double stranded DNA
2. This provides access for DNA replication machinery collectively referred to as the replisome

Question 22

What are the 2 phases (biphasic) of DNA replication initiation in eukaryotes?

Answer 22

1. Replicator selection occurs in G1 phases- formation of pre-replication complex
2. Origin activation occurs in S phase- unwinding of DNA and recruitment of DNA polymerase

Ensures that each origin is used and each chromosome is only replicated once per cell cycle.

Question 23

Describe eukaryotic replicator selection leading to the formation of the pre-replication complex

Answer 23

1. Origin recognition complex bind to the replicator sequence (eg ARS in yeast)
2. Helicase loading proteins Cdc6 and Cdt1 are recruited and bind to the ORC
3. ATP hydrolysis opens the two hexametric helicase MCM2-7 rings, allowing them to wrap around the DNA in a head-head orientation
4. This concludes the formation of the pre-replication complex

Question 24

What do high levels of Cdk activate and prevent during S-phase

Answer 24

Activates existing pre-RC complexes but prevents the formation of new pre-RC complexes

Question 25

What does the close relationship between pre-RC, Cdk levels and cell cycle ensure?

Answer 25

Ensures that chromosomes are replicated exactly once per cell cycle

Question 26

What does Ribonuclease H do?

Answer 26

Removes RNA primers, further shortening the newly synthesised DNA strands at the 5’ ends of chromosomes

Question 27

What is the role of telomerase at the end of DNA replication?

Answer 27

1. Adds TTAGGG repeats to the 3’ end of every replicated molecule of DNA
2. This prevents shortening so no coding information is lost

Question 28

What is the telomere shuffle?

Answer 28

Telomerase provides its own RNA template primer and can use reverse transcriptase activity to polymerise DNA from its RNA template