Lecture 12 - DNA replication

Question 1

What direction is DNA (or RNA) always synthesised?

Answer 1

5’ to 3’

Question 2

What way is the parental template strand run?

Answer 2

3’ to 5’

Question 3

Where do you find origins of replication and why?

Answer 3

At AT rich regions because A-T strands are easier to pull apart than G-C strands because A-T has two carbons as opposed to three.

Question 4

What are the steps needed to make a copy of DNA?

Answer 4

Progressive addition of new nucleotides

A starting point for nucleotide addition

Unwinding of the helical double stranded DNA

Release of tension generated by unwinding the DNA helix

Prevention of unwound double stranded helical DNA from reforming and to protect it

Joining ends of the newly synthesised fragments together

Question 5

Why is replication of DNA considered semi-discontinuous?

Answer 5

The leading strand is continuously synthesised in its 5’ to 3’ direction but the lagging strand is discontinuously synthesised in its 5’ to 3’ direction as Okazaki fragments

Question 6

What is the function of helicase?

Answer 6

To pull the two DNA strands apart. When this happens the strands get tighter

Question 7

What is primase and its function?

Answer 7

An enzyme (a type of RNA molecule) that makes an RNA primer = starting point for DNA polymerisation.

Question 8

What is DNA polymerase lll and it’s function

Answer 8

An enzyme that synthesises a new DNA strand by adding nucleotides complimentary to the parental template strands. It does this in the 5’ to 3’ direction.

Question 9

How are primase and DNA polymerase lll connected?

Answer 9

Pol lll needs an -OH group onto which the phosphate group of the incoming nucleotide can be attached. Primase has an internal -OH group which the strands can add to. Pol lll will add one nucleotide at a time to the RNA primer.

Question 10

What is the function of topoisomerase?

Answer 10

To cut the two DNA strand so they can unwind which releases tension. After it sticks the pieces together

Question 11

What is the function of DNA polymerase l?

Answer 11

To remove RNA primers (RNase H) and fills the gap with DNA nucleotides (DNA polymerase)

Question 12

How does DNA polymerase l complete its function?

Answer 12

DNA pol l can recognise DNA RNA hybrids and it removes the RNA nucleotides and uses the -OH of the next door fragments and extends to the other fragment (can’t join them but extends)

Question 13

What is the function of DNA ligase?

Answer 13

To join the newly synthesised Okazaki fragments together (creates phosphodiester bonds), once the RNA primers have been removed and replaced by DNA nucleotides

Also joins the newly synthesised fragments from multiple replication bubbles, including the leading strands.

Question 14

What two activities does DNA pol l carry out?

Answer 14

1. RNase activity: RNase H is an endonuclease enzyme that recognises DNA:RNA hybrids and degrades the RNA cell
2. DNA polymerase activity: synthesises DNA by adding nucleotides (complementary to the parental DNA template of the lagging strand)

Question 15

What is the order of enzymes used to make a DNA copy?

Answer 15

Helicase
Primase
DNA polymerase lll
Topoisomerase 
Single-stranded DNA binding proteins 
DNA polymerase l 
DNA ligase

Question 16

What is the function of the single-stranded DNA binding protein?

Answer 16

Prevent two parent strands from snapping back together

Single strand DNA will prevent DNA ends from being degraded

Question 17

When can DNA errors be repaired and with what?

Answer 17

During replication - using an exonuclease

After replication - using an endonuclease

Question 18

What does exonuclease do?

Answer 18

Cuts DNA from the inside

Question 19

What does endonuclease do?

Answer 19

Cuts DNA from the outside

Question 20

How does the cell check for DNA errors and fix them during DNA replication?

Answer 20

DNA pol lll has a proofreading mechanism that checks the newly inserted nucleotide bases against the template as they are being added. If there is an incorrect base, it is removed by a 3’ to 5’ exonuclease activity of DNA polymerase lll.

Question 21

What is the reason for DNA errors found after DNA replication and how is it fixed?

Answer 21

DNA errors found after DNA replication can be from:
Incorrectly inserted bases are not corrected by DNA pol lll
Radiation damage (e.g. UV)
Chemical modifications of bases (natural and chemical causes)

These types or errors are removed by an endonuclease

Question 22

Why is it important to correct DNA errors?

Answer 22

If it’s not corrected the DNA error can become part of the DNA template which means a permanent DNA change (DNA damage/mutation)

Question 23

What is in vitro DNA synthesis?

Answer 23

DNA replication in the test tube

Question 24

What is a polymerase chain reaction (PCR)?

Answer 24

an in vitro method of making multiple DNA copies so that there is enough DNA material to work with

Question 25

What happens during PCR?

Answer 25

Only ‘targeted’ DNA region will be copied

Rapid exponential increase of DNA molecules

Method utilises cycles of heating and cooling

Question 26

What is PCR used for?

Answer 26

Medical applications
Forensic applications
Infectious disease detection and identification
Molecular biology research applications

Question 27

What is In vivo DNA synthesis?

Answer 27

DNA synthesis in cells

Question 28

What is the function of DNA template in ‘in vitro’ DNA replication?

Answer 28

DNA molecule to which complementary nucleotides can be matched to make identical copies via DNA synthesis

Question 29

What is the function of primers in ‘in vitro’ DNA replication?

Answer 29

Provide a free ‘3 OH group, the chemical group that is essential to initiate DNA synthesis

Defines the region of the DNA molecule to be replicated

Question 30

What is the function of DNA polymerase in ‘in vitro’ DNA replication?

Answer 30

Enzyme which add nucleotides, (complementary to the DNA template), and joins them together forming a phosphodiester bond.

Question 31

What is the function of dNTPs in ‘in vitro’ DNA replication?

Answer 31

Free nucleotides (equal amount of A, G, C and G) - the building blocks used by the DNA polymerase

Question 32

Compare primers In vivo vs In vitro

Answer 32

In vivo: RNA primer, synthesised by primase, removed and replaced by DNA nucleotides

In vitro: DNA primer, added completed, part of newly-synthesised DNA

Question 33

Compare DNA strands In vivo vs In vitro

Answer 33

In vivo: one continuous strand (leading), and discontinuous strand (lagging)

In vitro: two continuous strands

Question 34

Compare start In vivo vs In vitro

Answer 34

In vivo: at ‘origin of replication’ sites (multiple ori sites in eukaryotes)

In vitro: two sites where DNA primers are designed to bind

Question 35

Compare synthesis In vivo vs In vitro

Answer 35

In vivo: 5’ to 3’ direction all DNA (whole genome) synthesised

In vitro: 5’ to 3’ direction. Region between the primers synthesised.

Question 36

Compare temperature In vivo vs In vitro

Answer 36

In vivo: one constant temperature -37C

In vitro: cycling of different temperatures (45-98C)

Question 37

Compare proteins and enzymes In vivo vs In vitro

Answer 37

In vivo: Several enzymes and proteins (DNA helicase, topoisomerase, single-stranded DNA binding proteins, primase, DNA pol lll, DNA pol l, DNA ligase)

In vitro: one enzyme - heat stable DNA polymerase adding nucleotides (all other steps are regulated by temperatures)