Enzymes Flashcards
What would be at the 5 and 3 end of a DNA molecule that has been exposed to a
nuclease?
5’: PO4
3’: -OH
Explain what it means when nucleases can be damage / structure / sequence specific.
- Damage: Scan for damage -repair damaged DNA (DNA pol. add new bases to site)
- Structure: (recognise) intermediates generated by DNA repair mechanisms
- Sequence: recognise sequence of DNA (& break P-O bond on or near it)
Sequence specific nucleases are also referred to as?
Restriction endonucleases
Differentiate between endonuclease and exonuclease enzymes
- Exonuclease: Cuts DNA from free ends; cuts ssDNA
- Endonuclease: cut in b/w (middle) DNA; cuts dsDNA
Which essential cofactor do nucleases require to work?
Mg2+
Differentiate between blunt ends and sticky ends.
- Blunt: no overhanging bases @ ends (P-O bonds broken)
- Sticky: overhanging bases @ ends (have non-paired nucleotides) (P-O bond & H-bond broken)
what is a 5’ or 3’ overhang is when referring to sticky ends.
5’ ————— 3’ (5’ no pair)
(5’ eg) 3’ ————- 5’
(3’ eg) 5’ ————–3’
3’ —————-5’ (3’ no pair)
What kind of enzyme is DNAase I? What is it used for?
Endonuclease. Remove DNA from solution (e.g. DNA footprinting analysis)
How does DNAseI behave with different cofactors?
- Mg2+: Cuts independently = random fragments (P-O bond cut randomly along strands of ds DNA)
- Mn2+: Cleaves at dsDNA leaving 1-2 nucleotide overhangs
Explain how ligases work, and how they are used during DNA replication.
form P-O bond b/w 5’ P & 3’ OH of 2 strands (blunt/sticky).»_space; join Okazaki fragments tog. in DNA replication
When would an exonuclease be used?
When you want to cut overhangs -> blunt ends
What circumstance would you want to use an endonuclease?
For Host Restriction Modification Systems- use restriction endonuclease to cut thru DNA @ specific sequence
Which catalytic activity of DNA pol I is not present in the Klenow fragment? And what’s the other 2 catalytic activity
5’ to 3’ exonuclease bc it acts as an invasion pol.- displace downstream bases as it fills dNTP in ss section in DNA.
- 5’ to 3’ polymerase = proofread
- 3’ to d’ exonuclease = cut/fill 3’ overhangs
Which polymerase does not require a template strand? What’s it used for & its requirements?
Terminal Deoxynucleotidyl Transferase.
> make 3’ overhangs (on ssDNA/dsDNA 3< bp)
- Mg2+ & dNTP
Explain how the bacterial restriction modification system protects the cell against viral DNA?
Restriction endonucl./enzyme (RE) defends cell from invading DNA. But Bact. DNA & phage DNA is similar recognition capacity, so to distinguish itself from viral DNA, methylase (M) adds methyl group to that capacity. If recog. site for RE is methylated =can’t cut DNA w/ methyl = “self’ DNA proteted. While invading DNA is cleaved bc not methylated
