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Flashcards in Enzymes Deck (33)
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What do nucleases do?

Cut, shorten, and degrade nucleic acid molecules


What do ligases do?

Join nucleic acid molecules together


What do polymerases do?

Make copies of nucleic acid molecules


What do modifying enzymes do?

Remove or add chemical groups to nucleic acid molecules


What do topoisomerases do?

Introduce or remove supercoils from covalently closed circular DNA


How do nucleases degrade DNA molecules?

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


What are the 2 kinds of nucleases?



Describe how exonucleases work

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


Describe how endonucleases work

Break internal phosphodiester bonds within a DNA molecule


What are the 2 kinds of RNase?

RNase A
RNase H


Describe how RNase A works

Endoribonuclease that specifically degrades ssRNA


Describe how RNase H works

Endoribonuclease that digests the RNA of an RNA-DNA hybrid


How do ligases work?

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


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

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


Describe how DNA polymerase I works

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


Where is DNA polymerase I most commonly used?

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


Describe how Klenow fragment DNA polymerase works

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


Where is Klenow fragment DNA polymerase most commonly used?

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


Describe how reverse transcriptase works

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


Where is reverse transcriptase most commonly used?

Synthesis of cDNA for cloning
Labelling the termini of DNA fragments with protruding 5' ends


Describe how Taq polymerase works

5-3' polymerase ONLY


Where is Taq polymerase most commonly used?

Used in PCR but requires specific primers


What are the 4 types of DNA modifying enzymes?

Alkaline phosphatase
Terminal deoxynucleotide transferase
DNA methylase
Polynucleotide kinase


Describe alkaline phosphatase

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


Describe terminal deoxynucleotidyl transferase

Adds >1 deoxynucleotide onto the 3' terminus of DNA
Used for the 3' tailing reaction


Describe DNA methylase

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


Describe polynucleotide kinase

Adds phosphate groups on to free 5' termini


What are the 3 classes of endonucleases?

Class I
Class II
Class III


What do class I endonucleases do?

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


What do class II endonucleases do?

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