Restriction enzymes

Question 1

Describe the lytic cycle.

Answer 1

• Single phage infects a single bacterium.
• The phage then reproduces to give 50-100 progeny.
• The infected cell lyses and the released phage can infect the surrounding cells.
• Phage then reproduce in surrounding cells.
• Infected cells lyse and the released phage can infect surrounding cells to produce a cleared area or plaque

Question 2

In a lab what conditions and amounts should be used for the lytic cycle to occur?

Answer 2

The bacteria should in high amounts compared to phage and they should be stored overnight at 37 degrees before plaques can be observed.

Question 3

What are plaques and how many phages are there in a single plaque?

Answer 3

Plaques are areas where bacteria have been killed.

In a single plaque there are 10^5 - 10^6 phages.

Question 4

What is the ‘lawn’?

Answer 4

Areas where there is bacterial growth.

Question 5

What are nucleases?

Answer 5

Enzymes that break phosphodiester bonds.

Question 6

What are the types of nucleases that show specificity to sugar

Answer 6

DNases, RNases and non specific nucleases.

Question 7

Other than sugar, how can nucleases differ?

Answer 7

They can be specific for only double stranded or single stranded or they can be non specific.
They can cut the phosphodiester bond in different places - either 5’ phosphate and 3’ hydroxyl or 5’ hydroxyl and 3’ phosphate.
They can differ by where they attack - endonucleases cleave site within the molecule and exonucleases degrade a molecule from one end.

Question 8

How do nuclease break the phosphodiester bond?

Answer 8

By hydrolysis - the addition of water.

Question 9

Where can the nucleases break the bond?

Answer 9

Either between the 3’ O and phosphate group or between the 5’ O and phosphate.

Question 10

Explain what the letters and numbers of EcoRI indicates.

Answer 10

First three letters indicates bacterial species of origin (genus + strain) Eco - Escherichia coli
The fourth letter indicates bacterial strain R - RY13
The Roman numerals differentiates between enzymes of this strain - I (first discovered).

Question 11

What is a palindrome?

Answer 11

A word or verse or sentence that reads the same backwards as it does forwards.

Question 12

Describe a mirror-like palindrome.

Answer 12

A sequence that reads the same forward and backwards on a SINGLE STRAND of DNA strand (e.g. GTAATG)

Question 13

Describe an inverted palindrome.

Answer 13

The sequence of top strand read LEFT to RIGHT (5’ to 3’) is same as the sequence of the bottom strand read RIGHT to LEFT (5’ to 3’) - more common in restriction enzymes.
5’ GAATTC 3’
3’ CTTAAG 5’

Question 14

How can DNA be protected from enzyme degradation?

Answer 14

Modification of DNA by methylation within the restriction enzyme recognition site.

Question 15

What does methylation of enzyme recognition site affect in DNA?

Answer 15

In changes the surface of major groove of DNA so that enzymes no longer recognise their target sequence.
No change in H bonding.

Question 16

Why have restriction enzymes evolved?

Answer 16

As a defence against bacterial viruses.

Question 17

How is a bacteria’s own DNA protected from digestion?

Answer 17

Via DNa modification normally methylation.

Question 18

What are the two properties of restriction enzymes that make them extremely useful?

Answer 18

There high specificity and precise cleavage.

- restriction enzymes always recognise exactly the same sequence and cut in the same position.

Question 19

Describe the structure of most restriction enzymes.

Answer 19

They are composed of two identical subunits (homodimer).
There are two binding sites present and both must interact with DNA for cleavage to occur.
Binding sites consist of two identical recognition sequences that are always located on the complimentary strands of the DNA and form a palindrome.

Question 20

Why might a sequence not be recognised when only read of a single strand of the double helix?

Answer 20

Because the sequence is a palindrome and this only becomes apparent when both the top and bottom strand have been read 5’ - 3’.

Question 21

How does a restriction enzyme recognise a palindrome?

Answer 21

One subunit recognises the sequence on the top strand and the other recognises the sequence on the bottom strand.

Question 22

Where does each subunit cleave?

Answer 22

The restriction enzymes cleaves both strands at the same position.

Question 23

What does the cleavage of each phosphodiester bond by the restriction enzyme result in?

Answer 23

The phosphate remaining on the 5’ end of DNA while the 3’ end is left with an OH group.
(note: could go other way but I think it is rare?)

Question 24

What does the cleavage of the two strands of DNA result in?

Answer 24

An overhang of (certain number of bases) at the 5’ end of the molecule these are termed sticky ends.

Question 25

Why are the overhangs termed ‘sticky ends’?

Answer 25

Because they easily pair with other fragments with complementary sticky ends.

Question 26

When are sticky ends not generated?

Answer 26

When cleavage occurs in the centre of a palindrome to produce blunt ends.

Question 27

Roughly how long are the sequences that the enzymes recognise?

Answer 27

Between 4-8 base pairs long.

Question 28

How is the frequency of cutting on a given DNA sequence affected by recognition sequence length?

Answer 28

The longer the sequence the lower the frequency of cutting in a DNA strand - the less of that sequence in the DNA.

Question 29

What is required to join two complementary sequences together (generated by same enzyme) and why?

Answer 29

DNA ligase is required because even though hydrogen bonds can form between complementary base pairs without ligase this is not strong enough. There must be a covalent phosphodiester bond form between the sugar phosphate backbone - this requires DNA ligase and ATP.

Question 30

What is bacterial DNA methylated by and how?

Answer 30

Methyltransferase - covalently bind a methyl group to nucleotide in DNA shortly after replication. This allows cell to distinguish between ‘self’ DNA and ‘non self’ DNA.

Question 31

On which bases and where does methylation occur?

Answer 31

Cytosine - at the N4 or C5 position

Adenine - at the N6 position

Question 32

During DNA replication what kind of bases are used by DNA polymerase?

Answer 32

Normal, non methylated bases.

Question 33

After replication of DNA will the DNA strands be methylated?

Answer 33

Only one strand in the duplex will be methylated (original strand) and the other will not be. This is called hemi-methylated.

Question 34

Is hemi-methylated DNA cleaved by restriction enzymes?

Answer 34

No.

Question 35

Why is hemi-methylated DNA converted to fully methylated DNA if it is not cleaved by restriction enzymes?

Answer 35

It is the best substrate for methylases and therefore is quickly fully methylated before the next round of replication.

Question 36

What happens when unmethylated DNA enters a cell?

Answer 36

The DNA will be subject to methylation by methylases however this is a very inefficient process so it most likely to be degraded by endonucleases which is a efficient process.

Question 37

Describe a type 1 restriction modification system.

Answer 37

Asymmetric discontinuous recognition site.
Methylate recognition site but cleave remotely.
Multisubunit protein complex usually containing 2 restriction endonuclease subunits (REase or restriction enzyme), 2 methyltransferase subunits (MTase or methylase) and 1 specificity subunit.
ATP required for cleavage.

Question 38

Describe a type 2 restriction modification system.

Answer 38

Symmetric recognition.
Cleave and methylate within or close to recognition site.
REase usually acts independently of MTase.
Act as monomer, dimer or even tetramers.
ATP not required for cleavage.

Question 39

Describe a type 3 restriction modification system.

Answer 39

Complex containing both REase subunit and MTase subunit.

ATP required for cleavage

Question 40

What is a isoenzyme?

Answer 40

enzymes that have the same recognition site but not necessarily the same cleavage site or sensitivity to methylation.