DNA Recplicaton

Question 1

Directions of DNA synthesis:

Answer 1

• DNA (or RNA) is synthesised in the 5’ —-> 3’ direction (remember the 3rd carbon OH group)
• thus parents template strands run the 3’ ——> direction

Question 2

Eukaryotic DNA replication

Answer 2

• Hydrogen bonds are broken at AT rich regions and synthesised from that single point in opposite directions
• multiple larger linear chromosomes, 23 pairs in humans
• multiple origins of replication (ori)
• bi directional

Question 3

What is needed to make a DNA copy?

Answer 3

• progressive addition of new nucleotides (A,C,T,G)
• a starting point for nucleotide addition
• unwinding of the helical double-stranded DNA (to give two parental templates)
• release of tension generated by unwinding the DNA helix
• prevention of unwound double-stranded helical DNA, i.e. single-stranded DNA, from reforming and to protect it
• joining of ends of newly synthesised fragments together (lagging as well as leading strands)

Question 4

Leading strand is…

Answer 4

Continuously synthesised in its 5’ —-> 3’ direction

Question 5

Lagging strand:

Answer 5

Discontinuously synthesised in it’s 5’ —-> 3’ direction as Okazaki fragments

Question 6

AT rich area

Answer 6

• helicase pulls strands apart - recognising rich regions
• as each is strand is synthesised - helicase continues to pull apart

Question 7

Primase

Answer 7

• an enzyme (type of RNA polymerase) that makes an RNA primer= starting point of DNA polymerisation

Question 8

DNA polymerase 111

Answer 8

• an enzyme that synthesises a new DNA strand by adding nucleotides complementary to parental template strands
• cannot bind to a single stranded DNA and start copying it - needs an OH group onto which the phosphate group of the incoming nucleotide can be attached
• 5’ to 3’ direction

Question 9

DNA pol 1 function

Answer 9

Removes RNA primers and fills the gap with DNA nucleotides

Question 10

DNA Ligase

Answer 10

1. Joins the newly synthesised Okazaki fragments together (creates phosphodiester binds), once the RNA primers have been removed and replaced by DNA nucleotides
2. Joined together the newly synthesised fragments form the multiple replication bubblers, including the leading strands.

Question 11

DNA pol 1 carries out 2 activities

Answer 11

1. RNase Activity:
   - RNase H is an endonuclease enzyme that recognises RNA:DNA hybrids and degreases the RNA part
2. DNA Polymerase Activity:
   - synthesises DNA by adding nucleotides ( complementary to the parental DNA template of the lagging strand)

Question 12

Topoisomerase

Answer 12

• cuts strands apart to release tension then glues them back together

Question 13

Single stranded binding proteins

Answer 13

• prevents strands from joining back together once separated
• Protects single strands of DNA from enzymes
• DNA pol 111 moves along and knocks off said proteins

Question 14

Responsible for progressive addition of new nucleotides (A,C,T,G)

Answer 14

DNA polymerase 111

Question 15

What enzyme is responsible for making the starting point for nucleotide addition

Answer 15

• primase enzyme makes RNA primer

Question 16

What unwinds the helical double stranded DNA to give two parental strands

Answer 16

Helicase

Question 17

Prevention of unwound double- stranded helical DNA, i.e. single-stranded DNA, from reforming and to protect it from degradation

Answer 17

Single-stranded DNA binding protein

Question 18

Removes RNA primer and fills gap with DNA nucleotides

Answer 18

DNA polymerase 1 (RNase H activity removes RNA and DNA polymerase adds the DNA nucleotides)

Question 19

What is responsible for joining newly synthesised fragments together (lagging as well as leading strands, within and between replication bubbles)

Answer 19

DNA ligase

Question 20

When can DNA errors be repaired?

Answer 20

1. During replication (using an EXOnuclease) - will move from end to end ( usually 3-5 sometimes 5-3)
2. After replication (using an ENDOnuclease) - can move within, doesn’t need an end - can remove from end

Question 21

Replication error rare of DNA pol 111

Answer 21

1 in 10^8 - 10^10 base-pairs replicated

(DNA pol 111 has a proofreading mechanism which checks the newly inserted nucleotide bases against the template)

Question 22

How are incorrect bases corrected during DNA synthesis

Answer 22

• through the 3’ to 5’ activity of DNA pol 111

-Damaged/ incorrect DNA
I
V
- incorrect base is removed by the 3’ to 5’ exonuclease activity of DNA pol 111
I
V
- DNA synthesis conitinues

Question 23

What can cause DNA errors or damage after DNA replication?

Answer 23

• incorrectly inserted bases are not corrected by DNA pol 111
• radiation damage (U.V)
• chemical modifications of base (natural and Chemical causes)

These types of incorrect or damaged nucleotide bases are removed by endonuclease

Question 24

How damaged nucleotide bases are removed by removing an endonuclease

Answer 24

• Damaged / incorrect DNA
• damage, including some surrounding (flaking) regions, is removed by endonuclease
• A DNA polymerase makes new DNA
• DNA ligaments joins new DNA to existing DNA

Question 25

What happens if the DNA is not corrected?

Answer 25

• DNA error becomes part of DNA template thus permemant DNA change (mutation)

DNA helix (where one of the strands carry’s an error) separated for the replication process what’re each DNA strand will be used as a template

The DNA strand being synthesised using incorrect DNA template will carry a permanent error. All future DNA molecules arising form this ‘mutated’ DNA will carry the incorrect DNA base.

Question 26

What happens in the polymerase chain reaction (PCR) (test tube)

Answer 26

• in vitro method of making multiple DNA copies do that there is enough DNA material to work with
• only ‘targeted’ DNA region will be copied
• rapid exponential increase of DNA molecules
• method utilises cycles of heating and cooling

Question 27

PCR can be used in:

Answer 27

• medical applications
• forensic applications
• infectious disease detection and identification
• molecular biology research and applications

Question 28

How PCR works:

Answer 28

1. Denaturation: is increased, denaturing hydrogen bonds and separating DNA strands
2. Annealing: temp is lowered to allow primers to base pair to complementary DNA template
3. Extension: polymerase extends primer to form nascent DNA strand

THIS PROCESS REPEATES UP TO 35 TIMES

1st cycle - 2^2
2nd - 3^2
3rd - 4^2

Question 29

Functions of PCR components unused ‘in vitro’ DNA replication

Answer 29

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

Primers:
- provides a free ‘OH group, the chemical group that is essential to initiate DNA synthesis
- defines the region of the DNA molecule to be replicated

DNA polymerase:
- enzyme which addds nucleotides, (complementary to the DNA template), and joins them together forming a phosphodiester bond

dNTPs:
- free nucleotides (equal amount of each) - the building b locked used by the DNA polymerase

Question 30

DN