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BIO2030 Biotechnology > Enzymes > Flashcards

Flashcards in Enzymes Deck (33)
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1
Q

What do nucleases do?

A

Cut, shorten, and degrade nucleic acid molecules

2
Q

What do ligases do?

A

Join nucleic acid molecules together

3
Q

What do polymerases do?

A

Make copies of nucleic acid molecules

4
Q

What do modifying enzymes do?

A

Remove or add chemical groups to nucleic acid molecules

5
Q

What do topoisomerases do?

A

Introduce or remove supercoils from covalently closed circular DNA

6
Q

How do nucleases degrade DNA molecules?

A

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

7
Q

What are the 2 kinds of nucleases?

A

Exonucleases

Endonucleases

8
Q

Describe how exonucleases work

A

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

9
Q

Describe how endonucleases work

A

Break internal phosphodiester bonds within a DNA molecule

10
Q

What are the 2 kinds of RNase?

A

RNase A

RNase H

11
Q

Describe how RNase A works

A

Endoribonuclease that specifically degrades ssRNA

12
Q

Describe how RNase H works

A

Endoribonuclease that digests the RNA of an RNA-DNA hybrid

13
Q

How do ligases work?

A

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

14
Q

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

A
4
DNA polymerase I
Klenow fragment DNA polymerase 
Reverse transcriptase (RNA-dependant DNA polymerase)
Taq polymerase
15
Q

Describe how DNA polymerase I works

A
Is a DNA dependant DNA polymerase 
Has 3 different modes of actions;
5'-3' polymerase 
5'3' exonuclease
3'-5' exonuclease
16
Q

Where is DNA polymerase I most commonly used?

A

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

17
Q

Describe how Klenow fragment DNA polymerase works

A
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
18
Q

Where is Klenow fragment DNA polymerase most commonly used?

A

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

19
Q

Describe how reverse transcriptase works

A
Is an RNA dependant DNA polymerase 
# different modes of action;
5'-3' polymerase 
5'-3' riboexonuclease 
3'-5' exoribonuclease
20
Q

Where is reverse transcriptase most commonly used?

A

Synthesis of cDNA for cloning

Labelling the termini of DNA fragments with protruding 5’ ends

21
Q

Describe how Taq polymerase works

A

5-3’ polymerase ONLY

22
Q

Where is Taq polymerase most commonly used?

A

Used in PCR but requires specific primers

23
Q

What are the 4 types of DNA modifying enzymes?

A

Alkaline phosphatase
Terminal deoxynucleotide transferase
DNA methylase
Polynucleotide kinase

24
Q

Describe alkaline phosphatase

A

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

25
Q

Describe terminal deoxynucleotidyl transferase

A

Adds >1 deoxynucleotide onto the 3’ terminus of DNA

Used for the 3’ tailing reaction

26
Q

Describe DNA methylase

A

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

27
Q

Describe polynucleotide kinase

A

Adds phosphate groups on to free 5’ termini

28
Q

What are the 3 classes of endonucleases?

A

Class I
Class II
Class III

29
Q

What do class I endonucleases do?

A

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

30
Q

What do class II endonucleases do?

A

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

31
Q

What do class III endonucleases do?

A

Contain nuclease and methylase activity

The recognition site is not symmetrical

32
Q

What is an isoschizomer?

A

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

33
Q

What is a neoschizomer?

A

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