DNA Replication

Question 1

When is DNA replicated? (What phase)

Answer 1

During Synthesis phase

Question 2

What are cyclins?

Answer 2

a family of regulatory proteins that control the progression of the cell cycle

Question 3

What are 3 prediction models for DNA replication?

Answer 3

Conservative, Semi-conservative and dispersive

Question 4

What are the 10 requirements for DNA replication?

Answer 4

• Template strand
• primase
• DNA polymerase
• helicase
• DNA gyrase
• DNA ligase
• primer with a free recessed 3’OH group
• all four dNTPs (Deoxynucleoside triphosphates)
• Origin of replication
• Single-strand DNA binding proteins

Question 5

What are 4 properties of DNA replication?

Answer 5

Enzyme - DNA polymerase
Template - ssDNA template
Biochemical mechanism of action - nucleophilic attack of 3’OH
Driving force - hydrolysis of ppi

Question 6

What is the polymerase reaction simplified?

Answer 6

Parental DNA + dNTPs – (DNA polymerase and hydrolysis reaction) –> Progeny DNA strand + ppi

Question 7

Explain the nucleophilic attack. What electrolyte is needed?

Answer 7

When the 3’ OH group of the new strand attacks the phosphate group of the incoming complementary dNTP, hydrolysis PPi creates a bond. Mg2+ is also required.

Question 8

How does the polymerase know which nucleotide to add next? Why is DNA directional?

Answer 8

Hydrogen bonds are required for 2 dNTPs to bind, therefore only certain bases can pair together. The incoming nucleotides are therefore selected by DNA polymerase by their ability to form Watson-crick base pairs.

For new DNA to be synthesised the 3’OH nucleophilic attack must occur, therefore it can only be made 5’ - 3’

Question 9

Summarise and draw out the polymerase reaction

Answer 9

3p on incoming nucleotide binds to new strand. Hydrologists occurs and 2 p is released and cleaved to drive the reaction forward.

Question 10

What factors contribute to the accuracy of DNA replication?

Answer 10

• balanced nucleotide levels
• polymerase mechanism
• polymerase proof-reading mechanism (1 mistake per millions bps)
• activity of DNA repair enzymes

Question 11

What is the origin of replication in bacteria and eukaryotes? Which is faster?

Answer 11

Origins are specific locations in the DNA.
bacteria: single origin of replication. 1000 bp/ second
eukaryotes: multiple origins. 10 - 100 bp/second

Question 12

Explain the direction of bacterial DNA replication?

Answer 12

assembled at the single origin of DNA replication in a characteristic location.

Question 13

What is Okazaki’s model of discontinuous replication? How are Okazaki fragments joined later? Draw a diagram.

Answer 13

Says since DNA must be replicated 5’ to 3’, we have a leading strand of continuous replication and a lagging strand where Okazaki fragments are made following the primer. Later joined together by DNA ligase

both move as the replication fork advances.

Question 14

What are RNA primers?

Answer 14

Primase adds bits of RNA to initiate the start of DNA replication. Multiple used in the lagging strand

Question 15

What are 3 functions of DNA polymerase? What is unique about E.coli DNA polymerase l?

Answer 15

• polymerisation 5’ - 3’ adding nucleotides
• exonuclease 3’ - 5’ removes nucleotides in that direction
• exonuclease 5’ - 3’ removes nucleotides in that direction

E.coli DNA polymerase l can do all 3 functions.

Question 16

Explain the exonuclease activities of E.coli DNA polymerase l.

Answer 16

• 3’ -5’ exonuclease function of proof-reading
• 5’ - 3’ exonuclease function of DNA excises nucleotides from single-stranded nicks and removes rna primers