Enzymes Flashcards
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?
Exonucleases
Endonucleases
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?
4 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
What do class III endonucleases do?
Contain nuclease and methylase activity
The recognition site is not symmetrical
What is an isoschizomer?
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
What is a neoschizomer?
Enzymes that recognise the same sequence but cleave at different positions from the prototype
