Enzymes

Question 1

What do nucleases do?

Answer 1

Cut, shorten, and degrade nucleic acid molecules

Question 2

What do ligases do?

Answer 2

Join nucleic acid molecules together

Question 3

What do polymerases do?

Answer 3

Make copies of nucleic acid molecules

Question 4

What do modifying enzymes do?

Answer 4

Remove or add chemical groups to nucleic acid molecules

Question 5

What do topoisomerases do?

Answer 5

Introduce or remove supercoils from covalently closed circular DNA

Question 6

How do nucleases degrade DNA molecules?

Answer 6

Break the phosphodiester bonds that link one nucleotide to the next in a DNA strand

Question 7

What are the 2 kinds of nucleases?

Answer 7

Exonucleases

Endonucleases

Question 8

Describe how exonucleases work

Answer 8

Remove nucleotides one at a time from the end of a DNA molecule

Question 9

Describe how endonucleases work

Answer 9

Break internal phosphodiester bonds within a DNA molecule

Question 10

What are the 2 kinds of RNase?

Answer 10

RNase A

RNase H

Question 11

Describe how RNase A works

Answer 11

Endoribonuclease that specifically degrades ssRNA

Question 12

Describe how RNase H works

Answer 12

Endoribonuclease that digests the RNA of an RNA-DNA hybrid

Question 13

How do ligases work?

Answer 13

Covalently links the free ends of DNA molecules and repairs the ss break in one of the strands of a ds molecule
Can also join together individual DNA molecules or the two cohesive ends of the same molecule
Catalyses the formation of a phosphodiester bond between adjacent 5’-P and 3’-OH termini in DNA
Can work on blunt or sticky ends
H bonding gives a stable structure for the enzyme to work on

Question 14

How many types of polymerase are there and what are they?

Answer 14

4
DNA polymerase I
Klenow fragment DNA polymerase 
Reverse transcriptase (RNA-dependant DNA polymerase)
Taq polymerase

Question 15

Describe how DNA polymerase I works

Answer 15

Is a DNA dependant DNA polymerase 
Has 3 different modes of actions;
5'-3' polymerase 
5'3' exonuclease
3'-5' exonuclease

Question 16

Where is DNA polymerase I most commonly used?

Answer 16

Nick translation
Probe preparation
Repairing DNA fragments
Producing blunt-end DNA from sticky-end DNA

Question 17

Describe how Klenow fragment DNA polymerase works

Answer 17

Is a DNA dependant DNA polymerase 
Has 2 different modes of action;
5'-3' polymerase 
3'-5' exonuclease 
Can only synthesise a complementary DNA strand on a single-stranded template

Question 18

Where is Klenow fragment DNA polymerase most commonly used?

Answer 18

Sanger dideoxy sequencing
Synthesis of a synthesis of cDNA in cDNA cloning
Filling in the 3’ recessed termini created by digestion of DNA with a restriction enzyme
Labelling of the termini of the DNA fragment

Question 19

Describe how reverse transcriptase works

Answer 19

Is an RNA dependant DNA polymerase 
# different modes of action;
5'-3' polymerase 
5'-3' riboexonuclease 
3'-5' exoribonuclease

Question 20

Where is reverse transcriptase most commonly used?

Answer 20

Synthesis of cDNA for cloning

Labelling the termini of DNA fragments with protruding 5’ ends

Question 21

Describe how Taq polymerase works

Answer 21

5-3’ polymerase ONLY

Question 22

Where is Taq polymerase most commonly used?

Answer 22

Used in PCR but requires specific primers

Question 23

What are the 4 types of DNA modifying enzymes?

Answer 23

Alkaline phosphatase
Terminal deoxynucleotide transferase
DNA methylase
Polynucleotide kinase

Question 24

Describe alkaline phosphatase

Answer 24

Removes a phosphate group at the 5’ terminus of a DNA molecule
Is used to prevent recircularisation of a plasmid during cloning work

Question 25

Describe terminal deoxynucleotidyl transferase

Answer 25

Adds >1 deoxynucleotide onto the 3’ terminus of DNA

Used for the 3’ tailing reaction

Question 26

Describe DNA methylase

Answer 26

Transfers a methyl group to internal A or C residues to produce methylated duplex DNA
Used to protect DNA from restriction enzymes

Question 27

Describe polynucleotide kinase

Answer 27

Adds phosphate groups on to free 5’ termini

Question 28

What are the 3 classes of endonucleases?

Answer 28

Class I
Class II
Class III

Question 29

What do class I endonucleases do?

Answer 29

Recognise some specific sequence but aren’t useful in gene manipulation since their cleavage site is non-specific

Question 30

What do class II endonucleases do?

Answer 30

Are Mg2+ dependant with a highly specific recognition site
Very useful for DNA manipulation
Are restriction enzymes
Used for cutting DNA in a very precise and reproducible way during molecular cloning work
They cut both strands of dsDNA within a palindromic recognition sequence
They hydrolyse the sugar-phosphate backbone to give a 5’- P on one side and a 3’-OH on the other
Give sticky or blunt ends

Question 31

What do class III endonucleases do?

Answer 31

Contain nuclease and methylase activity

The recognition site is not symmetrical

Question 32

What is an isoschizomer?

Answer 32

A restriction enzyme that recognises the same sequence as another restriction enzyme
The first example discovered is called the prototype and all subsequent enzymes are isoschizomers of the prototype

Question 33

What is a neoschizomer?

Answer 33

Enzymes that recognise the same sequence but cleave at different positions from the prototype